Small molecule conjugates specifically activated in tumor microenvironment for targeting and use thereof

ABSTRACT

Provided is an anticancer compound including a cleavable linker specifically activated in a tumor microenvironment, and use thereof. The anticancer compound is represented by the following formula, wherein, R 1  is a normal functional group or a protection group; R 2  is Ala, Thr, Val or Ile; R 3  is Ala, Val or Asn; R 4  is a drug group linked via a hydroxyl group or an amino group; and the general formula of the drug is R 4 H. The anticancer compound is only activated at a local portion of a tumor, thus avoiding the defect of immune system damage of a traditional chemotherapeutic drug, and promoting tumor immunization by removing a tumor immunosuppression cell. The anticancer compound or pharmaceutical composition thereof is jointly used with immunotherapy, thus improving the effect of treating the tumor, and effectively inhibiting tumor metastasis and osseous metastasis.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

Any and all applications for which a foreign or domestic priority claimis identified in the Application Data Sheet as filed with the presentapplication are hereby incorporated by reference under 37 CFR 1.57.

TECHNICAL FIELD

The present disclosure belongs to a field of pharmaceutical chemistry,relating to an anti-tumor drug compound. Specifically, the presentdisclosure relates to a cleavable linker specifically activated in tumormicroenvironment, an anti-tumor compound comprising a conjugate and usethereof.

TECHNICAL BACKGROUND

Conventional cytotoxic chemotherapy drugs have great toxicity to humannormal cells and immune system. For example, Docetaxel and Paclitaxelare effective anti-tumor agents widely used at present. They are mainlyused in various solid tumors, such as ovarian cancer and breast cancer,and have a certain efficacy against lung cancer, intestinal cancer,melanoma, head and neck cancer, lymphoma and cerebroma. Clinically,these two compounds have serious toxicity, such as arrest of bonemarrow, and allergic reaction, and thus their doses have beenrestricted. Docetaxel exhibits bone marrow toxicity, resulting inreduction in neutrophilic granulocytes, and neurotoxicity andcardiovascular toxicity. Docetaxel can induce an allergic reaction, anda local inflammation, alopecia, hypodynamia, or even liver toxicity ifit overflows the blood vessel. Mitomycin is another effective antitumoragent widely used at present. It is mainly used in various solid tumors,such as stomach cancer, colon cancer, liver cancer, pancreatic cancer,non-small cell lung cancer, breast cancer, and malignant ascitic fluid.However, clinically, mitomycin exhibits a serious toxicity and adversereaction, its dose thus is restricted. Mitomycin can induce a bonemarrow toxicity, resulting in reduction in leucocytes and platelets. Itcan also induce phlebitis, and tissue necrosis, alopecia, hypodynamiaand hepatorenal damage if it overflows the blood vessel.

In the tumor microenvironment, the tumor cells express and secrete agreat amount of asparagine endopeptidases. Expression of asparagineendopeptidase can distinguish the tumor-associated macrophage (M2 type)from the mononuclear cell and the inflammatory macrophage (M1 type). Thecytokines secreted by tumor induce the mononuclear cells to transform totumor-associated macrophages. The tumor-associated macrophages canstimulate and produce strong immunosuppression and directly helpinfiltration and metastasis of tumor cells. Meanwhile, a great amount ofproteolytic enzymes are produced during metastasis of tumor cells todegrade intercellular matrix. Thus, new compounds can be chemicallysynthesized and screened based on biochemistry and pharmacologicaldetection and screening to find a chemical conjugate that is able to beactivated by asparagine endopeptidase and conjugated to drug via asecondary activated linker. The conjugate can link different groups forsolubility or modification as needed to drugs for chemotherapy havingspecific cytotoxicity, thus producing new drugs having new functions,such as new targeting, activation, stability, solubility, metabolism,toxicity and efficacy, etc.

SUMMARY OF INVENTION

In order to develop antitumor drugs, the present disclosure creates acleavable linker having changeable properties, such as activation bytargetedly conjugating and treatment by dissolution, and providescompounds containing a cleavable linker, as shown in formulae (I) and(II). Use of the cleavable linker of the present disclosure, which canbe specially activated in a tumor microenvironment, can effectivelyblock the toxicity of the linked drug R₄. Then the compounds aretargetedly activated by an asparagine endopeptidase in the tumormicroenvironment and the 4-aminobenzyl-OC(O)— is self-released, allowingthe final drugs the bring about new targeting, activation and metabolismproperties.

Specifically, in the compounds containing a cleavable linker, thecleavable linker is the modified tripeptide in the brackets,—R₂-R₃-Asn-4-aminobenzyl-OC(O)—. R₁ and R₄ link together through thecleavable linker, wherein R₁ links to the cleavable linker through anamide bond formed by its carbonyl, and R₄ links to the cleavable linkerthrough carbonic acid ester bond formed by its oxygen atom with thecleavable linker or through carbamate formed by its nitrogen atom withthe cleavable linker:R₁{-R₂-R₃-Asn-4-amino benzyl-OC(O)—}R₄  (I),

wherein

-   -   R₁ is a functional group for increasing solubility or a        protective group;    -   R₂ is an amino acid moiety selected from the group consisting of        Ala, Thr, Val and Ile, R₂ forms an amide bond with R₁ through a        carbonyl group of;

R₃ is an amino acid moiety selected from the group consisting of Ala,Thr, Val and Asn;

R₂ links to R₃ through an amide bond, R₃ links to Asn through an amidebond, and Asn links to —NH— through its carbonyl;

R₄ is a drug group linking to the cleavable linker through carbonic acidester bond or carbamate formed by its hydroxyl or amino;

the compounds containing the cleavable linker can be cleavable bycontact with an asparagine endopeptidase and then is separated from R₁;and

breakage of the compound containing the cleavable linker by contact withan asparagine endopeptidase causes further cleavage of the carbonic acidester bond or carbamate formed with R₄, resulting in that R₄ isseparated from the cleavable group.

In the present disclosure, compounds containing activatable conjugateswere synthesized, the compounds (formula (I),(II)) containing acleavable linker have the following structure-efficacy relationship ofstructure and activation:

(1) The activatable conjugates can react with hydroxyl or amino grouphaving proper activation grade in the toxic function related key part ofR₄ via group conversion, and couple to form drugs with new structure.The resultant compounds have different activation efficiencies byasparagine endopeptidase due to their different steric hindrances. Thisis because the enzyme active center of asparagine endopeptidase locatesat the bottom of its globular depression. The cleavage site needs toapproach the active center. Thus, the polarity of the linking sitebecomes important as it will determine whether a steric hindrance to thecleavage site is produced by the linked compound. The extension andsecondary breakage of 4-amino benzyl —OC(O)— arm effectively reduce thesteric hindrance from some drugs. However, in the present disclosure S6,S20 and some unpublished compounds cannot be activated because of sterichindrance. Although they are synthesized, they cannot form functionalcompounds containing a cleavable linker (2) Through specific activationby asparagine endopeptidase specially expressed by tumor cells or tumorassociated macrophages, the compounds containing the cleavable linkerare locally activated in the tumor and thus have a targetedcytotoxicity. The drugs which could not be activated due to sterichindrance are not toxic or have low toxicity to the cells, and theycannot form anti-tumor drugs. (3) Cytotoxicity of drugs decreasesgreatly after connecting the conjugate, because the conjugate reactswith hydroxyl or amino group of drugs, and the active hydroxyl or aminogroups on cell surface are usually key groups for drug cytotoxicity. (4)The compounds containing the cleavable linker are stable in non-tumorousenvironment, such as in blood, normal organs, immune system and inneutral pH, and have no toxic or low toxicity. (5) The asparagineendopeptidase cleaves the conjugate at the Asn site. According to theanalysis on the metabolites, only breakage can initiate the activationbetween 4-amino benzyl —OC(O)— and R₄, and thus assisting the cascadeactivation. (6) As a linking arm, 4-amino benzyl —OC(O)— can extend thelinkage, and thus can effectively reduce the steric hindrance close tothe reactive center of the asparagine endopeptidase after linking to R₄.However, the activation by contacting with asparagine endopeptidase isstill affected by the structure and polarity of R₄. (7) The polarity,solubility coming with R₄ are related to the activation efficiency ofthe conjugate, and are closely relevant to the solubility, stability andefficacy of the drugs containing the cleavable linker. In addition tothe conventional linking group, R₁ can link to a special hydrophilicgroup or targeted group to bring a special function for the drugscontaining the cleavable linker, such as improvement of solubility, andefficacy in the Examples. (8) In line with the distribution of theasparagine endopeptidase, the compounds containing the cleavable linkercan be activated in many kinds of tumors. They can broaden the scope ofthe diseases to be treated by the drug due to the changed solubility.Therefore, antitumor drugs against various tumor or a specific tumor canbe developed. (9) During metastasis of tumor cells, a great amount ofasparagine endopeptidases are secreted by the cells to degradeintercellular matrix. Therefore, the targeted drugs after linking to thecleavable linker exhibit a special efficacy to tumor metastasis. (10)The compounds containing the cleavable linker have low toxicity and highefficacy, they are nontoxic to immune system and can be combined withimmunotherapy at the same time, making a synergistic efficacy.

Description of the compounds and examples are as follows:

(1) Compounds S1˜S43, S15′, B15 and E15: Demonstrating new anti-cancercompounds can be synthesized by linking to the cleavable linker via twotypes of connection (Example 1-9), different anti-cancer compound showssynthetic efficiency and toxicity reduction (Example 9), differentactivation efficiencies (Example 10, 11) and efficacy (Example 12),providing comparative studies for the cleavable linker when usedtogether with different R₁, R₂, R₃ (Example 13˜14).

(2) Compounds S2′ ˜S4′ and S10′ ˜S24′, and compounds A1, A3˜A4 andA10˜A24: indicating that Docetaxel compounds containing the cleavablelinker and different R₁, R₂ and R₃ can be synthesized (Example 16, 17,27 and 28). The linking site, steric hindrance, connection length andvariation of R₁ bring about different solubility (Example 17 and 28,improved solubility), different activation efficiency (Example 20 and30), low toxicity, high efficacy and new indications (Example 20-26,31-35 and 66).

(3) Compounds B1, B3˜B4 and B10˜B24, compounds D2˜D4 and D10˜D24:indicating that Docetaxel compounds containing the cleavable linker anddifferent R₁, R₂ and R₃ can be synthesized (Example 36, 37, 46 and 47).The linking site, steric hindrance, connection length and variation ofR₁ bring about different solubility (Example 37 and 47, improvedsolubility), different activation efficiency (Example 38 and 49), lowtoxicity, high efficacy and new indications (Example 40-45 and 50-54).

(4) Compounds E2˜E4 and E10˜E24: indicating that Mitomycin compoundscontaining the cleavable linker and different R₁, R₂ and R₃ can besynthesized (Example 56 and 57). The linking site, steric hindrance,connection length and variation of R₁ bring about different solubility(Example 57, improved solubility), different activation efficiency(Example 58), low toxicity, high efficacy and new indications (Example59-65).

In one embodiment, the compound of formula (II) has a structure as setforth in any of the following formulae (IIA), (IIB), (IIC), (IID),(III), (IV), (V), (VI), (VII), (VIII), and (IX):

wherein

R₁ is selected from the group consisting of 6-maleimide-C₁₋₁₀alkylcarbonyl, hydroxylaminocarbonyl-C₁₋₁₀ alkylcarbonyl, C₁₋₄alkoxyl-(C₁₋₄ alkoxyl)_(n)-C₁₋₆ alkylcarbonyl, or

wherein each R is independently a C₁₋₄alkyl, and each n is independentlyany integer between 1-300, preferably 1-150;

R₂ is Ala, Thr, Val or Ile;

R₃ is Ala, Thr, Val or Asn;

R₅ is the active moiety of an anticancer compound containing a hydroxylgroup (R₅—OH), i.e., a moiety except the hydroxyl group used forlinking, wherein the anticancer compound is selected from the groupconsisting of Camptothecin, 10-Hydroxyl Camptothecin, Topotecan,Floxuridine, 5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide,Fludarabine, Capecitabine, Vincristine, Epothilone B, Paclitaxel andDocetaxel; and

R₆ is the active moiety of an anticancer compound containing an aminogroup (R₅—NH₂), i.e., a moiety except the amino group used for linking,wherein the anticancer compound is selected from the group consisting ofDaunorubicin, Epirubicin, Methotrexate, Fludarabine, Gemcitabine,Cytarabine, MelphalaN, Nimustine, Mitoxantrone and Mitomycin.

The present disclosure also provides a pharmaceutical compositioncomprising the compound of formula (II) or a pharmaceutically acceptablesalt thereof and a pharmaceutically acceptable carrier or excipient.

The present disclosure provides a method for preparing a compound offormula (III) or (IV), which is shown as follows:

wherein the preparation of the compound of formula (III) comprisesreacting R₁-R₂-R₃-Asn-4-amino benzyl alcohol with 4-nitrophenylchloroformate or (CCl₃O)₂CO to form an active carbonic acid ester bondor chloroformate, and then reacting the active carbonic acid ester bondor chloroformate with the drug comprising a hydroxyl group (R₅—OH) toform the compound of formula (III), wherein the drug is selected fromthe group consisting of Camptothecin, 10-Hydroxyl Camptothecin,Topotecan, Floxuridine, 5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide,Fludarabine, Capecitabine, Vincristine, Epothilone B, Paclitaxel andDocetaxel;

the preparation of the compound of formula (IV) comprises reactingR₁-R₂-R₃-Asn-4-amino benzyl alcohol with 4-nitrophenyl chloroformate or(CCl₃O)₂CO to form an active carbonic acid ester bond or chloroformate,and then reacting the active carbonic acid ester bond or chloroformatewith the drug comprising an amino group (R₆—NH₂) to form the compound offormula (IV), wherein the drug is selected from the group consisting ofDaunorubicin, Epirubicin, Methotrexate, Fludarabine, Gemcitabine,Cytarabine, Melphalan, Nimustine, Mitoxantrone and Mitomycin;

wherein R₁ is a conventional functional group or a protecting group; R₂is Ala, Thr, Val or Ile; and R₃ is Ala, Thr, Val or Asn.

The present disclosure provides use of the compound of formula (II) or apharmaceutically acceptable salt thereof or the pharmaceuticalcomposition of the present disclosure in the manufacture of a medicamentfor treating or preventing a cancer.

The present disclosure provides use of a mitomycin derivative as shownin formula (IX) or a pharmaceutically acceptable salt thereof in themanufacture of a medicament for treating or preventing an ophthalmicdisease.

The present disclosure provides use of the compound of formula (II) or apharmaceutically acceptable salt thereof or the pharmaceuticalcomposition of the present disclosure in the manufacture of a medicamentfor inhibiting tumor-associated macrophages, tumor growth, angiogenesisor infiltration and metastasis of tumor cells, and/or promotinganti-tumor immunization.

The present disclosure also provides a method for treating or preventinga cancer, comprising administering a subject in need thereof atherapeutically or prophylactically effective amount of the compound offormula (II) or a pharmaceutically acceptable salt thereof or thepharmaceutical composition of the present disclosure.

The present disclosure also provides a method for reducing the toxicityof an anticancer compound, comprising linking the anticancer compound toR₁-R₂-R₃, wherein R₄ is a conventional functional group or a protectinggroup; R₂ is Ala, Thr, Val or Ile; R₃ is Ala, Thr, Val or Asn; and theanticancer compound is selected from the group consisting ofCamptothecin, 10-Hydroxyl Camptothecin, Topotecan, Floxuridine,5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide, Fludarabine,Capecitabine, Vincristine, Epothilone B, Daunorubicin, Epirubicin,Methotrexate, Gemcitabine, Melphalan, Nimustine, Mitoxantrone,Paclitaxel, Docetaxel and Mitomycin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show comparative experiments performed in HT1080 modelby using a high dose of Legutaxel, Capxol and Paclitaxel injections,which were used at an equal molar dose and at an equal toxic dose.

FIG. 2 shows the experimental results obtained from immunologicalstimulation test for Paclitaxel and Legutaxel, demonstrating that moretoxic CD8 T cells (shown by the arrows in the right panel) werepermeated from the tumor tissue treated by Lagutaxel.

FIGS. 3A and 3B show the experimental results obtained fromimmunological stimulation test for Paclitaxel and Legutaxel.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT I. Compounds

The compounds of the present disclosure comprises conjugates as shown informula (A), and compounds of formula (II) formed by conjugating theconjugates to a drug R₄. The compounds of formula (II) could accumulateat the tumor site, and are specifically activated, thus releasing theantitumor compound.

The compound of formula (A) of the present disclosure has a structure asset forth below:

wherein, R₁ is a conventional functional group or a protecting group; R₂is Ala, Thr, Val or Ile; R₃ is Ala, Thr, Val or Asn; and R₇ is H,XC(O)—, or optionally substituted benzyloxycarbonyl (for example,optionally substituted by 1, 2 or 3 substituents selected from the groupconsisting of nitro, C₁₋₄ alkyl, halogen, hydroxyl and amino), wherein Xis halogen; wherein R₁ links to R₂ through an amide bond formed by thecarbonyl of R₁; and amide bonds are formed between R₂ and R₃, R₃ andAsn, and Asn and —NH—.

The present disclosure also provides a compound of formula (II):

wherein R₁ is a conventional functional group or a protecting group; R₂is Ala, Thr, Val or Ile; R₃ is Ala, Thr, Val or Asn; and R₄ is an activemoiety of a drug linking through hydroxyl or amino group, and the drugis represented by formula R₄—H.

In some embodiments, in the compounds of the present disclosure, R₁links to R₂ by forming an amide bond via its carbonyl, R₂, R₃ and Asnform a tripeptide, Asn links to —NH— via its carbonyl, R₄ links to thecleavable linker through carbonic acid ester bond formed by its oxygenatom with the cleavable linker or through carbamate formed by itsnitrogen atom with the cleavable linker.

In some preferred embodiments, R₄ links to the cleavable linker througha carbamate formed by its nitrogen atom of the amino substituent on thearomatic ring with the cleavable linker, or through carbonic acid esterbond formed by its oxygen atom of hydroxyl substituent on the aromaticring or heterocycle with the cleavable linker.

In some embodiments, R₃ is preferably Ala.

In the present disclosure, R₁ can be H or an amino protecting group. Forexample, R₁ can be a hydrophilic or hydrophobic group. Alternatively, R₁can be selected from any of C₁₋₆ alkyl (such as methyl, ethyl, propyl,butyl, pentyl, hexyl), polyethylene glycol-C₁₋₅ alkylcarbonyl, succinyl,glucosiduronide, maleimide-C₁₋₁₀ alkylcarbonyl (such as 6-maleimidecaproyl), 2-methoxyethoxy-C₁₋₆ alkylcarbonyl,hydroxylaminocarbonyl-C₁₋₁₀ alkylcarbonyl (such asN-hydroxylamino-1,8-octandioic acid-1-monoacyl) and caproyl (C₁₋₅alkylcarbonyl).

Preferably, R₁ can be 6-maleimide-C₁₋₁₀ alkylcarbonyl,hydroxylaminocarbonyl-C₁₋₁₀ alkylcarbonyl, C₁₋₄ alkoxyl-(C₁₋₄alkoxyl)_(n)-C₁₋₆ alkylcarbonyl, or

wherein each R is independently a C₁₋₄ alkyl, and each n isindependently any integer between 1-300, preferably 1-150.

Preferably, when R₄—H is a water-insoluble drug, R₁ is preferably aPEG-type group, such as polyethylene glycol-C₁-C₅ alkylcarbonyl, or

Generally, R₁ links to the amino group of R₂, and when R₁ links to R₂via its carbonyl, an amide linkage (—CO—NH—) forms.

In the present disclosure, R₄ is the active moiety of an anticancercompound, wherein the anticancer compound includes, but is not limitedto, Camptothecin, 10-Hydroxyl Camptothecin, Topotecan, Floxuridine,5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide, Fludarabine,Capecitabine, Vincristine, Epothilone B, Daunorubicin, Epirubicin,Methotrexate, Gemcitabine, Melphalan, Nimustine, Mitoxantrone,Paclitaxel, Docetaxel and Mitomycin.

The compounds of formula (II) may include the compounds having any ofthe following structures:

In one embodiment, R₄ is —O—R₅, and the compound of formula (II) has astructure set forth in the following formula (III):

wherein R₅ is the active moiety of an anticancer compound containing ahydroxyl group (R₅—OH), i.e., a moiety except the hydroxyl group usedfor linking, wherein the anticancer compound is selected from the groupconsisting of Camptothecin, 10-Hydroxyl Camptothecin, Topotecan,Floxuridine, 5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide,Fludarabine, Capecitabine, Vincristine, Epothilone B, Paclitaxel andDocetaxel.

In one embodiment, R₄ is R₆—NH, and the compound of formula (II) has astructure set forth in the following formula (IV):

wherein R₆ is the active moiety of an anticancer compound containing anamino group (R₆—NH₂), i.e., a moiety except the amino group used forlinking, wherein the anticancer compound is selected from the groupconsisting of Daunorubicin, Epirubicin, Methotrexate, Fludarabine,Gemcitabine, Cytarabine, Melphalan, Nimustine, Mitoxantrone andMitomycin. In formula (IV), “(H)” represents that H is present or notpresent; if H is not present, N links to R₆ via a double bond.

In each structure of the present disclosure, n generally is an integerin the range of 1-300, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 . . . 100,101, 102, 103 . . . 201, 202, 203 . . . 295, 296, 297, 298, 299 and 300.It should be understood that, although each integer between 1-300 is notspecifically described, these un-described integers are obvious to theskilled artisan, and it should be constructed as that the presentdisclosure has literally disclosed all of the integers falling withinthe range. In the present disclosure, n in each structure generally isin the range of 1-250, 1-200, 1-150, 1-100, 1-50, and such as 5-10,5-50, 5-100 and the like.

In one embodiment, compounds of formula (III) include:

-   -   (1) Compound S1 in which R₁ is 2-(2-methoxyethoxy) acetyl, R₂ is        Thr, R₃ is Ala, and R₄ is 10-hydroxyl camptothecin

-   -   (2) Compound S2 in which R₁ is 2-(2-methoxyethoxy) acetyl, R₂ is        Ala, R₃ is Ala, and R₄ is camptothecin

-   -   (3) Compound S3 in which R₁ is (N-hydroxylamino)-1,8-octandioic        acid-1-monoacyl, R₂ is Ala, R₃ is Ala, and R₄ is Capecitabine

and

-   -   (4) Compounds S7-S18, wherein R₁ is 2-(2-Methoxyethoxy)Acetyl,        R₂ is Thr, R₃ is Ala and R₄ is Camptothecin (S7), 10-Hydroxyl        Camptothecin (S8), Topotecan (S9), Floxuridine (S10),        5′-Deoxy-5-Fluorouridine (S11), Cytarabine (S12), Fludarabine        (S13), Etoposide (S14), Capecitabine (S15), Gemcitabine (S16),        Vincristine (S17), or Epothilone B (S18). The compounds and the        position of the hydroxyl are as follows:

camptothecin-cleavable linker which is specifically activated in thetumor microenvironment

10-hydroxyl camptothecin-cleavable linker which is specificallyactivated in the tumor microenvironment

topotecan-cleavable linker which is specifically activated in the tumormicroenvironment

floxuridine-cleavable linker which is specifically activated in thetumor microenvironment

5′-Deoxy-5-Fluorouridine-cleavable linker which is specificallyactivated in the tumor microenvironment

Cytarabine-cleavable linker which is specifically activated in the tumormicroenvironment

Fludarabine-cleavable linker which is specifically activated in thetumor microenvironment

Etoposide-cleavable linker which is specifically activated in the tumormicroenvironment

capecitabine-cleavable linker which is specifically activated in thetumor microenvironment

Gemcitabine-cleavable linker which is specifically activated in thetumor microenvironment

Vincristine-cleavable linker which is specifically activated in thetumor microenvironment

Epothilone B-cleavable linker which is specifically activated in thetumor microenvironment

In one embodiment, compounds of formula (IV) include:

-   -   (1) Compound S4 in which R₁ is 2-(2-methoxyethoxy) acetyl, R₂ is        Thr, R₃ is Ala, and R₄ is Daunorubicin

-   -   (2) Compound S5 in which R₁ is 6-maleimide caproyl, R₂ is Ala,        R₃ is Ala, and R₄ is Daunorubicin

and

-   -   (3) Compounds S19-S28, wherein R₁ is 2-(2-Methoxyethoxy)Acetyl,        R₂ and R₃ are Ala, R₄ is Daunorubicin (S19), Epirubicin (S20),        Fludarabine (S21), Gemcitabine (S22), Nimustine (S23),        Mitoxantrone (S24), Methotrexate (S25), Cytarabine (S26),        Melphalan (S27) or Doxorubicin (S28). The compounds and the        position of the amino group used for linking are as follows:

daunorubicin-cleavable linker which is specifically activated in thetumor microenvironment

Epirubicin-cleavable linker which is specifically activated in the tumormicroenvironment

Fludarabine-cleavable linker which is specifically activated in thetumor microenvironment

Gemcitabine-cleavable linker which is specifically activated in thetumor microenvironment

Nimustine-cleavable linker which is specifically activated in the tumormicroenvironment

Mitoxantrone-cleavable linker which is specifically activated in thetumor microenvironment

Methotrexate-cleavable linker which is specifically activated in thetumor microenvironment

Cytarabine-cleavable linker which is specifically activated in the tumormicroenvironment

Melphalan-cleavable linker which is specifically activated in the tumormicroenvironment

Doxorubcin-cleavable linker which is specifically activated in the tumormicroenvironment

In one embodiment, the present disclosure provides a paclitaxelderivative for targeted activation in the tumor microenvironment, whichhas a structure as set forth in the following formula (V):

wherein R₂ is Ala, Thr, Val or Ile; R₃ is Ala, Thr, Val or Asn; n is anyinteger between 1-300, preferably between 1-150.

Compounds of formula (V) include but is not limited to the followingcompounds:

-   -   (1) Compound S1′ in which n is 1, R₂ is Ala and R₃ is Ala

-   -   (2) Compound S2′ in which n is 5, R₂ is Ala and R₃ is Ala

-   -   (3) Compound S3′ in which n=11, R₂ is Ala and R₃ is Ala

-   -   (4) Compound S4′ in which n=300, R₂ is Ala and R₃ is Ala

and

-   -   (5) Compounds S10′-S24′ in which n is 1 and R₂ and R₃ are shown        in the following table:

No. of Compound R₂ R₃ n S10′ Ala Thr 1 S11′ Ala Val 1 S12′ Ala Asn 1S13′ Thr Ala 1 S14′ Thr Thr 1 S15′ Thr Val 1 S16′ Thr Asn 1 S17′ Val Ala1 S18′ Val Thr 1 S19′ Val Val 1 S20′ Val Asn 1 S21′ Ile Ala 1 S22′ IleThr 1 S23′ Ile Val 1 S24′ Ile Asn 1

In one embodiment, the present disclosure provides a water-solublepaclitaxel derivative for targeted activation, which has a structure asset forth in the following formula (VI):

wherein R₂ is Ala, Thr, Val or Ile, R₃ is Ala, Thr, Val or Asn, and n isany integer between 1-300, preferably between 1-150.

Compounds of formula (VI) include but is not limited to the followingcompounds:

-   -   (1) Compound A1 in which n is 1, R₂ is Ala and R₃ is Ala

-   -   (2) Compound A2 in which n is 5, R₂ is Ala and R₃ is Ala

-   -   (3) Compound A3 in which n is 11, R₂ is Ala and R₃ is Ala

-   -   (4) Compound A4 in which n is 150, R₂ is Ala and R₃ is Ala

Other compounds of formula (IV) include the following compounds, inwhich n is 5 and R₂ and R₃ are shown in the following table:

No. of Compound R₂ R₃ A10 Ala Thr A11 Ala Val A12 Ala Asn A13 Thr AlaA14 Thr Thr A15 Thr Val A16 Thr Asn A17 Val Ala A18 Val Thr A19 Val ValA20 Val Asn A21 Ile Ala A22 Ile Thr A23 Ile Val A24 Ile Asn

The present disclosure further provides a water-soluble Docetaxelderivative for targeted activation of tumor, which has a structure asset forth in the following formula (VII):

wherein R₂ is any one amino acid selected from the group consisting ofAla, Thr, Val and Ile; R₃ is any one amino acid selected from the groupconsisting of Ala, Thr, Val and Asn; n is any integer between 1-300,preferably between 1-150.

Compounds of formula (VII) include but are not limited to the followingcompounds:

-   -   (1) Compound B1 in which n is 1, R₂ is Ala and R₃ is Ala

-   -   (2) Compound B2 in which n is 5, R₂ is Ala and R₃ is Ala

-   -   (3) Compound B3 in which n is 11, R₂ is Ala and R₃ is Ala

-   -   (4) Compound B4 in which n is 150, R₂ is Ala and R₃ is Ala

Compounds of formula (VII) further comprise the following compounds:

No. of Compound R₂ R₃ n B10 Ala Thr 5 B11 Ala Val 5 B12 Ala Asn 5 B13Thr Ala 5 B14 Thr Thr 5 B15 Thr Val 5 B16 Thr Asn 5 B17 Val Ala 5 B18Val Thr 5 B19 Val Val 5 B20 Val Asn 5 B21 Ile Ala 5 B22 Ile Thr 5 B23Ile Val 5 B24 Ile Asn 5

The present disclosure further provides a Docetaxel derivative fortargeted activation of tumor microenvironment, which has a structure asset forth in the following formula (VIII):

wherein R₂ is any one amino acid selected from the group consisting ofAla, Thr, Val and Ile; R₃ is any one amino acid selected from the groupconsisting of Ala, Thr, Val and Asn; n is any integer between 1-300,preferably between 1-150, more preferably between 1-20, and mostpreferably between 1-11.

Compounds of formula (VIII) include:

-   -   (1) Compound D1, in which n is 1, R₂ and R₃ are Ala

-   -   (2) Compound D2, in which n is 5, R₂ and R₃ are Ala

-   -   (3) Compound D3, in which n is 11, R₂ and R₃ are Ala

-   -   (4) Compound D4, in which n is 300, R₂ and R₃ are Ala

Other compounds of formula (VIII) comprise the following compounds:

No. of Compound R₂ R₃ n D10 Ala Thr 1 D11 Ala Val 1 D12 Ala Asn 1 D13Thr Ala 1 D14 Thr Thr 1 D15 Thr Val 1 D16 Thr Asn 1 D17 Val Ala 1 D18Val Thr 1 D19 Val Val 1 D20 Val Asn 1 D21 Ile Ala 1 D22 Ile Thr 1 D23Ile Val 1 D24 Ile Asn 1

In one embodiment, the present disclosure provides a mitomycinderivative for release by targeted activation having a structure shownin the following formula (IX):

wherein R₂ is any amino acid selected from the group consisting of Ala,Thr, Val and Ile; R₃ is any amino acid selected from the groupconsisting of Ala, Thr, Val and Asn; n is any integer between 1-300,preferably between 1-150, more preferably between 1-20, and mostpreferably between 1-11.

Examples of compounds of formula (IX) include:

-   -   (1) Compound E1, in which n is 1 and R₂ and R₃ are Ala

-   -   (2) Compound E2, in which n is 5 and R₂ and R₃ are Ala

-   -   (3) Compound E3, in which n is 11 and R₂ and R₃ are Ala

-   -   (4) Compound E4, in which n is 300 and R₂ and R₃ are Ala

Examples of compounds of formula (XI) further comprise:

No. of Compound R₂ R₃ n E10 Ala Thr 1 E11 Ala Val 1 E12 Ala Asn 1 E13Thr Ala 1 E14 Thr Thr 1 E15 Thr Val 1 E16 Thr Asn 1 E17 Val Ala 1 E18Val Thr 1 E19 Val Val 1 E20 Val Asn 1 E21 Ile Ala 1 E22 Ile Thr 1 E23Ile Val 1 E24 Ile Asn 1

The pharmaceutically acceptable salt of the above compounds are alsoincluded in the present disclosure. Examples of pharmaceuticallyacceptable salts include inorganic and organic acid salts, such ashydrochloride, hydrobromide, phosphate, sulphate, citrate, lactate,tartrate, maleate, fumarate, mandelate and oxalate; and inorganic andorganic base salts with bases, such as sodium hydroxy, Tris(hydroxymethyl) aminomethane (TRIS, tromethamine) andN-methyl-glucamine.

II. Preparation of Compounds

R₁-R₂-R₃-Asn-4-amino benzyl alcohol is used as the key intermediate inthe present disclosure to prepare the present compounds. Preferably, thereaction schemes for preparing the present compound are as follows:

In Scheme 1, after reacting R₁-R₂-R₃-Asn-4-amino benzyl alcohol with4-nitrophenyl chloroformate or (CCl₃O)₂CO to form an active carbonicacid ester bond or chloroformate, the active carbonic acid ester bond orchloroformate is reacted with the drug comprising a hydroxyl group(R₅—OH) to form a carbonic acid diester product, which is also aconjugate. Scheme 1 can be used to prepare compounds in which R₄ isCamptothecin, 10-Hydroxyl Camptothecin, Topotecan, Floxuridine,5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide, Fludarabine,Capecitabine, Vincristine, Epothilone B, Paclitaxel or Docetaxel.

In Scheme 2, after reacting R₄-R₂-R₃-Asn-4-amino benzyl alcohol with4-nitrophenyl chloroformate or (CCl₃O)₂CO to form an active carbonicacid ester bond or chloroformate, the active carbonic acid ester bond orchloroformate is reacted with the drug comprising an amino group(R₆—NH₂) to form a carbonic acid diester product, which is also aconjugate. Scheme 2 can be used to prepare compounds in which R₄ isDaunorubicin, Epirubicin, Methotrexate, Fludarabine, Gemcitabine,Cytarabine, Melphalan, Nimustine, Mitoxantrone or Mitomycin.

Other reagents, reaction conditions, purification methods, etc., used inthe above preparation methods will be apparent to the skilled artisanafter reading the preparation examples disclosed herein.

III. Pharmaceutical Composition

The present disclosure comprises a pharmaceutical composition comprisinga compound of any of the above structural formulae or a pharmaceuticallyacceptable salt thereof.

The pharmaceutical composition may further comprise a pharmaceuticallyacceptable carrier or excipient. The carrier or excipient may be variouspharmaceutically acceptable carrier or excipient known in the art andcan be varied according to the dosage form or administration route.

In one embodiment, the pharmaceutical composition may comprise one ormore of solvents, solubilizer/co-solvent, pH adjustor, freeze-driedexcipient and osmo-regulator.

Freeze-dried excipient suitable for use in the present disclosureincludes one or more of sugars, such as lactose, maltose, dextran,glucose and fructose; amino acids, such as arginine, lysine and histine;mannitol; tartaric acid; maleic acid; citric acid; sodium chloride; andcyclodextrin, such as hydroxypropyl beta cyclodextrin and sulfobutylbeta cyclodextrin.

pH regulator suitable for use in the present disclosure includes one ormore of hydrochloric acid, phosphoric acid, sulfuric acid, carbonicacid, nitric acid, acetic acid, citric acid, DL-tartaric acid,D-tartaric acid, L-tartaric acid, NaOH, KOH, meglumine, maleic acid,ethylene diamine, triethylamine, arginine, lysine, histine, NaH₂PO₄ andNa₂HPO₄.

Solvent suitable for use in the present disclosure preferably is anorganic solvent, including one or more of ethanol, propylene glycol,polyethylene glycol 300, polyethylene glycol 400, t-butyl alcohol,glycerin, Tween, soybean oil, hydroxylpropyl beta cyclodextrin solutionand sulfobutyl beta cyclodextrin solution.

Osmo-regulator suitable for use in the present disclosure includes oneor more of glucose, sodium chloride, mannitol and sodium lactate.

Solubilizer/co-solvent suitable for use in the present disclosureincludes one or more of Tween 80, Tween 60, poloxamer, hydroxypropylbeta cyclodextrin, polyethylene glycol (PEG), lithium 12-hydroxystearate, sulfobutyl beta cyclodextrin, PVP, glycerin andpolyoxyethylene castor oil.

Typically, the compound of the present disclosure or itspharmaceutically acceptable salt thereof may be administered to mammals,orally at a dose of 0.0025 to 50 mg/kg of body weight, per day,preferably, approximately 0.01 to approximately 10 mg/kg of body weight.If a known anticancer agent or other treatments are also administered,they are administered in an amount that is effective to achieve theirintended purpose. The amounts of such known anticancer agents effectivefor cancer are well known to those skilled in the art.

The unit oral dose may comprise from approximately 0.01 mg toapproximately 50 mg, preferably approximately 0.1 mg to approximately 10mg of the compound of the invention or its pharmaceutically acceptablesalt. The unit dose may be administered one or more times daily, as oneor more tablets, each containing from approximately 0.1 mg toapproximately 50 mg, conveniently approximately 0.25 mg to 10 mg of thecompound or its pharmaceutically acceptable salt.

The pharmaceutical composition of the present disclosure may beformulated into any suitable dosage forms, including but is not limitedto tablet, capsule and injection, etc. The pharmaceutical composition ofthe present disclosure may be administered via known routes in the art,including oral administration, intravenous injection, intramuscularinjection, etc.

IV. Use of Compound and Pharmaceutical Composition

Cytokines secreted by tumor induce mononuclear cells to transform totumor associated macrophages (TAM). Tumor associated macrophage could bestimulated to product strong immunosuppression and could directly helpthe tumor cells to infiltrate and metastasize. Expression of asparagineendopeptidase can distinguish the tumor-associated macrophage (M2 type)from the mononuclear cell and the inflammatory macrophage (M1 type). Thecompounds of the subject invention can be activated to release in thepresence of asparagine endopeptidase. Since different moieties in theconjugate specifically activated by asparagine endopeptidase couldgreatly affect the targeting, activation, stability, toxicity andefficacy and the like of the final drug, using the conjugatespecifically activated by asparagine endopeptidase of the presentdisclosure could effectively reduce the toxicity of the linked drug,bring new targeting, activation and metabolism properties for the finaldrug, increase treatment effect on tumor, produce new adaptive tumordiseases and prevent tumor from metastasis. Thus, new structure andfunction could be produced.

It is also found in the present disclosure that the conjugatesreleasable in the tumor microenvironment, such as compounds of formulae(III) to (IX) could kill tumor associated macrophage, weakenimmunosuppressive cytokines in the microenvironment, and promote releaseof toxic CD8 cells to improve the immunization. More importantly, thesecompounds releasable in the tumor microenvironment could only beactivated in the tumor site, which is different from the traditionalchemotherapeutic drugs which impair the whole immune system. In theexperiments, the compounds releasable in the tumor microenvironment andprogrammed death-1 (PD-1) inhibitory antibody (PDL1 antibody, which iscommercially available and considered as a candidate havingimmunological treatment effect at present) show strong synergistictreatment and thus could solve the problem that immunological treatmentis difficult to be used in combination with chemotherapeutic drug.

Therefore, the compound, its pharmaceutically acceptable salt or thepharmaceutical composition of the present disclosure could be used totreat or prevent various diseases that were known to be treated byCamptothecin, 10-Hydroxyl Camptothecin, Topotecan, Floxuridine,5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide, Fludarabine,Capecitabine, Vincristine, Epothilone B, Paclitaxel, Docetaxel,Daunorubicin, Epirubicin, Methotrexate, Gemcitabine, Melphalan,Nimustine, Mitoxantrone, or Mitomycin, including cancer and ophthalmicdiseases.

For example, it is known in the art that camptothecin can be used totreat or prevent malignant tumor, psoriasis, wart, acute/chronicleukaemia and hepatosplenomegaly caused by schistosomiasis; 10-hydroxylcamptothecin can be used to treat or prevent stomach cancer, livercancer, head and neck cancer and leukaemia, etc.; paclitaxel is mainlyused to treat ovarian cancer and breast cancer, and is also effective intreating lung cancer, intestinal cancer, melanoma, head and neck cancer,lymphoma, brain cancer, etc; and mitomycin can be used to chroniclymphoma, chronic myeloid leukemia, esophageal carcinoma, stomachcancer, colon cancer, rectal cancer, lung cancer, pancreatic cancer,liver cancer, cervical cancer, cancer of the uterus, ovarian cancer,breast cancer, tumor at head and neck, bladder tumor and malignantcavity effusion, etc.

Therefore, for example, diseases that can be treated or prevented by thecompound, its pharmaceutically acceptable salt or the pharmaceuticalcomposition of the present disclosure include but is not limited tocancer in bladder, brain, breast/mammary gland, cervix, colon-rectum,oesophagus, kidney, liver, lung, nasopharynx, pancreas, prostate, skin,stomach, uterus, ovary, testicle and blood. Specifically, the cancerincludes bladder cancer, brain cancer, breast cancer or mammary cancer,cervical cancer, colon-rectal cancer, esophageal carcinoma, renalcancer, liver cancer, lung cancer, nasopharyngeal carcinoma, pancreaticcancer, prostate cancer, skin cancer, stomach cancer, uterus cancer,ovarian cancer, testicular cancer and blood cancer.

In one specific embodiment, the mitomycin derivative as shown in formula(IX) or a pharmaceutically acceptable thereof of the present disclosurecan be used to treat or prevent an ophthalmic disease, includingtreating or preventing scar after healing, choroidal neovascularization,or inhibiting macrophage. In other examples, the mitomycin derivative asshown in formula (IX) can also be used to treat or prevent cornealtransplantation, glaucoma, sequelae of pterygium surgery, etc.

The compound or pharmaceutical composition of the present disclosure canalso be used to prevent tumor metastasis, especially metastasis of tumorto lung. In one example, the compound or pharmaceutical composition ofthe present disclosure can be used to prevent metastasis of mammarycancer to lung.

Therefore, the present disclosure comprises a method for treating orpreventing a disease, comprising administering a subject in need thereofa therapeutically or prophylactically effective amount of the compoundof the present disclosure or a pharmaceutically acceptable salt thereof,or the pharmaceutical composition comprising the compound of the presentdisclosure or a pharmaceutically acceptable salt thereof.

The present disclosure also comprises a method for preventing tumormetastasis, comprising administering a subject in need thereof thecompound of the present disclosure or a pharmaceutically acceptable saltthereof, or the pharmaceutical composition comprising the compound ofthe present disclosure or a pharmaceutically acceptable salt thereof.Prevention of tumor metastasis comprising preventing tumor formetastasizing to lung and/or bone.

Tumor associated macrophage (TAM) is a key inflammatory cell, playingcrucial role in tumor associated inflammation. In the tumormicroenvironment, TAM promotes tumor development through affectingvarious biological properties of tumor. It secretes some molecules, suchas EGF, to directly promote growth of tumor cell and angiogenesis,thereby promoting tumor infiltration and metastasis and inhibitingfunctionating of acquired immunity. Accordingly, the present inventioncomprises a method for inhibiting tumor associated macrophage,comprising administering a subject in need thereof the compound or apharmaceutically acceptable salt thereof of the present disclosure, orthe pharmaceutical composition comprising the compound or apharmaceutically acceptable salt thereof of the present disclosure. Byinhibiting tumor associated macrophage, tumor growth, angiogenesis,infiltration and metastasis of cancer cell can be inhibited, andanti-tumor immunization can be promoted, thus cancer can be treatedand/or prevented. In one specific embodiment, the tumor associatedmacrophage expresses aspartate endopeptidase and is a M2 type cell.

The above-mentioned methods of the present disclosure can be used incombination with any radiotherapy or immunotherapy known in the art.

Therefore, the present disclosure also comprises compounds, theirpharmaceutically acceptable salts or pharmaceutical composition of thepresent disclosure useful in the above-mentioned methods and uses.

The present disclosure also comprises use of the compound of the presentdisclosure, its pharmaceutically acceptable salt or the pharmaceuticalcomposition of the present disclosure in the manufacture of a medicamentfor treating or preventing the above disease, such as cancer and cancermetastasis. The present disclosure also comprises use of the compound ofthe present disclosure or a pharmaceutically acceptable salt thereof orthe pharmaceutical composition of the present disclosure in themanufacture of a medicament for inhibiting tumor-associated macrophages,tumor growth, angiogenesis or infiltration and metastasis of tumorcells, or promoting anti-tumor immunization.

The present disclosure further provides a method for reducing thetoxicity of an anticancer compound (R₄—H), comprising linking theanticancer compound to R₁-R₂-R₃, wherein R₁, R₂ and R₃ are defined asabove.

The method for treatment or prevention of the present disclosurecomprises administering the compound or pharmaceutical composition ofthe present disclosure to the subject in need thereof. Administrationroute includes but is not limited to oral administration, intravenousinject, and intramuscular injection, etc. Subject includes mammal,especially human.

It should be understood that the “comprise” and “include” used hereinalso include “consist of”. The sum of all weight percentages or volumepercentages should be equal to 100%. Unless specifically indicated,various reagents and products used in the Examples are commerciallyavailable. And unless specifically indicated, the methods are performedaccording to the conventional techniques. The following Examples are notintended to limit the scope of the present disclosure.

V. Examples

The technical solutions of the present disclosure are furtherillustrated in connection with the following examples.

Example 1: Synthesis of Chemical Intermediates 1) Synthesis ofN—(N-benzyloxycarbonyl-L-alanyl)-L-Ala methyl ester (I)

N-benzyloxycarbonyl-L Ala (100 g, 0.45 mol) were dissolved inN,N-dimethylformamide (3 L). 1-hydroxylbenzotriazole (HOBt, 72.6 g, 0.54mol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride(EDC, 103.3 g, 0.54 mol) were added when stirring. After reacting for 1hour under stirring, the mixture was cooled to 0□ in an ice bath andL-Ala methyl ester (46.2 g, 0.45 mol) and N,N-diisopropylethylamine(173.8 g, 1.34 mol) in the N,N-dimethylformamide solution (1 L) wasdropped into the mixture. After dropping, the mixture was stirred underambient temperature for 10 hours. The solvents were removed byevaporation under reduced pressure. The crude product was dissolved indichloromethane (2 L) and washed subsequently by saturated ammoniumchloride solution, water and saturated sodium chloride solution. Theorganic phase was dried by anhydrous sodium sulphate. After removing thesolvents by evaporation under reduced pressure, the crude product wasrecrystallized to obtain a white solid I (101 g, Yield 73.1%).

2) Synthesis of N—(N-benzyloxycarbonyl-L-alanyl)-L-Ala (II)

N—(N-benzyloxycarbonyl-L-alanyl)-L-Ala methyl ester (100 g, 0.34 mol)were dissolved in a mixed solution of tetrahydrofuran (2 L) and water (1L). The mixture was cooled to 0□ and 1M lithium hydroxide solution (400mL) were dropped into the mixture. The resultant mixture was stirred forreaction for 10 hours. Concentrated hydrochloric acid was dropped toadjust the pH to be less than 6. Most of tetrahydrofuran were removed byrotary evaporation. The residual water phase was extracted bydichloromethane (1 L×3). The organic phase was dried by anhydrous sodiumsulphate. A white solid II was obtained after vaporizing and dryingunder reduced pressure (88 g; Yield, 92.2%).

3) Synthesis of4-N—(N-fluorenylmethoxycarbonyl-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (III)

N-fluorenylmethoxycarbonyl-N′-triphenylmethyl-L-asparagine (20 g, 0.03mol), 2-(7-azabenzotriazol)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (15 g, 0.04 mol), N,N-dimethylformamide (DMF)(200 mL) were added into a three-neck flask and stirred for 30 minutes.A solution of 4-amino benzyl alcohol (4.1 g, 0.03 mol) in DMF (5 mL),and N,N-diisopropyl ethylamine (DIPEA) (8.7 g, 0.06 mol) were addedseparately under 0° C. and the mixture was stirred at ambienttemperature for 3 hours. Most DMF were removed by rotary evaporation.The residue was dissolved in ethyl acetate (200 mL), washed subsequentlyby saturated ammonium chloride solution and saturated sodium chloridesolution and dried by anhydrous sodium sulphate. After filtration, thesolvent was removed by evaporation. The resultant crude product waspulping to obtain a white solid III (21.3 g, Yield 90%).

4) Synthesis of 4-N—(N′-triphenylmethyl-L-asparaginyl)-amino benzylalcohol (IV)

4-N—(N-fluorenylmethoxycarbonyl-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (13.0 g, 18 mmol) were dissolved in N,N-dimethylformamide(80 mL). Piperidine (30 mL) was added and then stirred at ambienttemperature for 2 hours. The solvents were removed by evaporation underreduced pressure. And the resultant product was dried under high vacuumwithin a vacuum drying oven to remove a small quantity of piperidine. Apale yellow solid IV was obtained, which could be use in the next stepwithout purification.

5) Synthesis of4-N—(N—(N—(N-benzyloxycarbonyl-L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (V)

N—(N-benzyloxycarbonyl-L-alanyl)-L-Ala (6.0 g, 20.4 mmol),benzotriazol-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU,11.6 g, 30.6 mmol) and DMF (50 mL) were added into a three-neck flaskand stirred for 30 minutes in an ice bath. A solution of4-N—(N′-triphenylmethyl-L-asparaginyl)-amino benzyl alcohol in DMF (50mL), and N,N-diisopropylethylamine (7.89 g, 61.2 mmol) were addedseparately under 0° C. The resultant mixture was stirred overnight atambient temperature. The solvents were removed by evaporation underreduced pressure. The residue was dissolved in ethyl acetate (200 mL),washed subsequently by saturated ammonium chloride solution andsaturated sodium chloride solution and dried by anhydrous sodiumsulphate. After filtration, the solvent was removed by evaporation. Theresultant crude product was recrystallized to obtain a white solid V (15g, Yield 97%).

6) Synthesis of4-N—(N-(L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (VI)

4-N—(N—(N—(N-benzyloxycarbonyl-L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (5.0 g, 6.61 mmol) were dissolved in THF (150 mL). 10%Pd/C (1 g) was added. After introducing hydrogen gas, the resultantmixture was stirred for reaction under normal temperature and normalpressure for 5 hours. Pd/C was removed by filtration and washed bymethanol. The filtrates and the washing solutions were pooled. Mostsolvents were removed by rotary evaporation to obtain a crude product.After column chromatography, a white solid VI was obtained (2.0 g, Yield49%).

7) Synthesis of 4-N—(N—N—(N-2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (VII)

2-(2-methoxyethoxy) acetic acid (432 mg, 3.22 mmol) were dissolved inN,N-dimethylformamide (20 mL). Benzotriazol-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (1.83 g, 4.83 mmol) were added and stirred for 30minutes. Then4-N—(N-(L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (2.0 g, 3.22 mmol) and N,N-diisopropylethylamine (1.24 g,9.61 mmol) in N,N-dimethylformamide (20 mL) were dropped into theresultant mixture. After dropping, the temperature was slowly raised toambient temperature and then the mixture was stirred for 10 hours. Mostof DMF were removed by evaporation under reduced pressure. The residuewas dissolved in acetyl acetate (200 mL), washed subsequently bysaturated ammonium chloride solution and saturated sodium chloridesolution and dried by anhydrous sodium sulphate. After filtration, thesolvent was removed by rotary evaporation. The resultant crude productwas purified by silica gel column chromatography to obtain a white solidVII (1.2 g, Yield 50%).

8) Synthesis of 4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzyl alcohol (VIII)

4-N—(N—N—(N-2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (VII) (1.0 g, 1.36 mmol) were dissolved indichloromethane (10 mL). Trifluoroacetic acid (2 mL) were added and thenthe resultant mixture was stirred at ambient temperature for 5 hours.The reaction solution was washed by water and separated. The organicphase was dried by anhydrous sodium sulphate and the solvents wereremoved by evaporation under reduced pressure. The residualtrifluoroacetic acid was removed by evaporation under high vacuum. Theresultant crude product was purified by column chromatography to obtainVIII (600 mg, Yield 89%).

9) Synthesis of 4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester

4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzyl alcohol (500 mg,1.01 mmol) were dissolved in dichloromethane (10 mL). The resultantmixture was cooled to 5□. p-nitrophenyl chloroformate (406 mg, 2.02mmol) in a dichloromethane solution and pyridine (160 mg, 2.03 mmol)were subsequently dropped into the mixture under protection by nitrogengas. After dropping, the resultant mixture was stirred at ambienttemperature overnight. The reaction solution was washed by water andseparated. The organic phase was dried by anhydrous sodium sulphate andthe solvents were removed by rotary evaporation. The resultant crudeproduct was purified by column chromatography to obtain a pale yellowsolid (450 mg, Yield 67%).

Example 2: Synthesis of 4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-threonyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-10-hydroxyl camptothecin-carbonic acid diester (S1)

4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester (330 mg, 0.5 mmol) and10-hydroxyl camptothecin (182 mg, 0.5 mmol) were dissolved in anhydrousN,N-dimethylformamide (10 mL). The resultant mixture was cooled to 0□and then 4-dimethyl pyridine (DMAP) (122 mg, 1.0 mmol) and 1-hydroxylbenzotriazole (27 mg, 0.2 mmol) were added. The resultant mixture wasstirred at ambient temperature overnight. The reaction solution waspoured into acetyl acetate (100 mL), washed subsequently by water (50mL×3) and saturated sodium chloride (50 mL), and dried by anhydroussodium sulphate. The solvents were removed by rotary evaporation toobtain a crude product. The crude product was purified by columnchromatography to obtain the target product S1, which is a pale yellowsolid (82 mg, Yield 19%).

Example 3: Synthesis of 4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-camptothecin-carbonic acid diester (S2)

Triphosgene (600 mg, 2.02 mmol) were dissolved in anhydrousdichloromethane (10 mL). The resultant mixture was cooled to −10□ orbelow. 4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzyl alcohol (500 mg,1.01 mmol) and pyridine (0.35 mL, 12.12 mmol) in dichloromethane (10 mL)were dropped into the mixture under protection by nitrogen gas and theresultant mixture was stirred at 0□ for 1 hour. The temperature of themixture was allowed to warm up to ambient temperature naturally. Afterstirring for 2 hours, camptothecin (348 mg, 1 mmol) in dichloromethane(10 mL) were dropped into the mixture. Reaction was taken place atambient temperature for 6 hours. The reaction solution was washedsubsequently by water (30 mL), saturated sodium bicarbonate solution (20mL) and saturated sodium chloride (20 mL), and dried by anhydrous sodiumsulphate and then by evaporation under reduced pressure. The residue waspurified by column chromatography to obtain a white solid (291 mg, Yield53.5%).

Example 4: Synthesis of 4-N—(N—(N—(N-(8-(N-hydroxylamino)-1,8-octandioicacid-1-monoacyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-capecitabine-carbonic acid diester (S3)

4-N—(N—(N—(N-(8-(N-hydroxylamino)-1,8-octandioicacid-1-monoacyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester (715 mg, 1.0 mmol) andcapecitabine (360 mg, 1.0 mmol) were dissolved by anhydrousN,N-dimethylformamide (20 mL) and cooled to 0□ or below. Then DMAP (244mg, 2.0 mmol) and 1-hydroxylbenzotriazole (27 mg, 0.2 mmol) were added.The resultant mixture was stirred at ambient temperature overnight. Thereaction solution was poured into acetyl acetate (100 mL), washedsubsequently by water (100 mL×3) and saturated sodium chloride (100 mL),and dried by anhydrous sodium sulphate. The solvents were removed byrotary evaporation to obtain a crude product. The crude product waspurified by column chromatography to obtain the target product S3, whichis a pale yellow solid (198 mg, Yield 21%).

Example 5: Synthesis of 4-N—(N—(N—(N-2-(2-methoxyethoxy)acetyl-L-threonyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-daunorubicin-carbamate (S4)

4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-threonyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester (264 mg, 0.4 mmol) andN,N-diisopropylethylamine (1 mL) were dissolved in N,N-dimethylformamide(10 mL). Daunorubicin (211 mg, 0.4 mmol) in a N,N-dimethylformamide (10mL) solution were dropped into the resultant mixture at 20□. Afterdropping, reaction was allowed to take place at ambient temperature for3 hours. The reaction solution was poured into methyl tert-butyl ether,stirred for 0.5 hour and then filtered. The resultant red solid waspurified by column chromatography to obtain a rid solid product S4 (177mg, Yield 42.2%).

Example 6: Synthesis of Compound S5 1) Synthesis of4-N—(N—(N—(N-(6-maleimidocaproyl-L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol

6-Maleimide caproic acid (120 mg, 0.57 mmol) were dissolved inN,N-dimethylformamide (20 mL). 1-hydroxylbenzotriazole (92 mg, 0.68mmol) and N,N-diisopropyl ethylamine (0.19 mL, 1.15 mmol) were added.4-N—(N—(N-(L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (353 mg, 0.57 mmol) was added into under protection bynitrogen gas. The resultant mixture was stirred for 0.5 hour and thencooled to 0□ in an ice bath. Then 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (120 mg, 0.62 mmol) in aN,N-dimethylformamide (10 mL) solution were dropped into the mixture.After dropping, the resultant mixture was warmed up to ambienttemperature and then stirred overnight. The reaction solution was pouredinto ethyl acetate (150 mL), and washed subsequently by water (100mL×3), 5% dilute hydrochloric acid (50 mL) and 5% sodium carbonate (50mL). The organic phase was dried by anhydrous sodium sulphate and thenby evaporation under reduced pressure. The resultant product waspurified by column chromatography to obtain the product, which is awhite solid (300 mg, Yield 64.8%).

2) Synthesis of 4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzyl alcohol

4-N—(N—(N—(N-(6-maleimidocaproyl-L-alanyl)-L-alanyl)-N′-triphenylmethyl-L-asparaginyl)-aminobenzyl alcohol (163 mg, 0.2 mmol) were dissolved in dichloromethane (5mL). Trifluoroacetic acid (2 mL) were added. The resultant mixture wasstirred at ambient temperature for 5 hours. The reaction solution waswashed by water and then separated. The organic phase was dried byanhydrous sodium sulphate and the solvents were removed by evaporationunder reduced pressure. The residual trifluoroacetic acid was removed byevaporation under high vacuum. The resultant crude product was purifiedby column chromatography to obtain a pale yellow solid (97 mg, Yield85%).

3) Synthesis of 4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester

4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzyl alcohol (814mg, 1.0 mmol) were dissolved in dichloromethane (100 mL) and cooled to0□ in an ice bath. p-nitrophenyl chloroformate (406 mg, 2.0 mmol) in adichloromethane solution (20 mL) and pyridine (160 mg, 2.0 mmol) weresubsequently dropped into the resultant mixture under protection bynitrogen gas. After dropping, the resultant mixture was warmed up toambient temperature and then stirred overnight. The reaction solutionwas washed by water and separated. The organic phase was dried byanhydrous sodium sulphate and the solvents were removed by evaporationunder reduced pressure. The resultant crude product was purified bycolumn chromatography to obtain a white solid (597 mg, Yield 81%).

4) Synthesis of 4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-daunorubicin-carbamate (S5)

4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester (200 mg, 0.27 mmol) weredissolved in N,N-dimethylformamide (30 mL). Daunorubicin hydrochloride(152 mg, 0.27 mmol) were added. The resultant mixture was cooled to 5□and then N,N-diisopropyl ethylamine (0.1 mL, 0.6 mmol) inN,N-dimethylformamide (2 mL) solution were dropped into the mixtureunder protection by nitrogen gas. After dropping, the mixture was warmedup to ambient temperature and stirred for reaction overnight. Thereaction solution was poured into methyl tert-butyl ether (600 mL),stirred for 0.5 hour and then filtered. The resultant red solid waspurified by column chromatography to obtain a rid solid product S5 (164mg, Yield 54%).

Example 7: Synthesis of 4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-MMAE-carbamate (S6)

4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester (298 mg, 0.40 mmol) weredissolved in N,N-dimethylformamide (30 mL). MMAE (monomethyl auristatin)hydrochloride (305 mg, 0.40 mmol) were added. The resultant mixture wascooled to 5□ and then N,N-diisopropyl ethylamine (0.1 mL, 0.6 mmol) inN,N-dimethylformamide (2 mL) solution under protection by nitrogen gas.After dropping, the mixture was warmed up to ambient temperature andstirred for reaction overnight. The reaction solution was poured intomethyl tert-butyl ether (600 mL), stirred for 0.5 hour and thenfiltered. The resultant red solid was purified by column chromatographyto obtain a rid solid product S6 (434 mg, Yield 82.4%).

The synthetic results of compounds 51, S2, S3, S4 and S5 are summarizedin Table 1. The mass-to-charge ratios of 51, S2, S3, S4 and S5 detectedby mass spectrum (MS) are 916, 885, 880, 1079 and 1513, respectively,which are consistent to their calculated mass-to-charge ratios, as shownin Table 1.

TABLE 1 the properties and MS data of S1-S5 No. R₁ R₁ R₂ R₃ R₄ MS dataCharacter S1

Hydrophilic group Thr Ala 10-hydroxyl camptothecin  916 Pale yellowpowder S2

Hydrophilic group Ala Ala camptothecin  885 White solid S3

Targeted group Ala Ala capecitabine  880 Pale yellow soild S4

Hydrophilic group Thr Ala daunorubicin 1079 Red powder S5

Targeted group Ala Ala daunorubicin 1513 Red powder

Using different R₂ and R₃ merely results in the use of differentstarting materials when linking the amino acid. Different side chains ofthe amino acid R₂ and R₃ did not influence the synthesis. Consistentwith the above methods, merely the corresponding R₂ amino acid and R₃amino acid were used in the synthesis. The reaction for linking R₄ wasalso the same as the method mentioned above, except that the catalyticconditions and the reaction drugs were different in the tumormicroenvironment.

Example 8

Conditions for linking the linking group for targeting a small moleculethat is specifically activated in the tumor microenvironment todifferent R₄ compounds are different.

1) In the above compounds, method for linking R₄ via hydroxyl isdifferent from the method for linking R₄ via amino.

Whether R₁R₂R₃-Asn-amino benzyl alcohol-p-nitrophenol-carbonic aciddiester could be successfully linked to R₆ via amino depends onselection of R₆. For example, the reaction between R₁R₂R₃-Asn-aminobenzyl alcohol-p-nitrophenol-carbonic acid diester and camptothecin isdifferent from the reaction with MMAE, mainly in that the reaction withMMAE is taken place via the strong nucleophilicity of the amino group ofMMAE (82.4%), while reaction of camptothecin is taken place viareplacement of p-nitrophenol through nucleophilicity of the hydroxylgroup of camptothecin. The nucleophilicity of hydroxyl is weaker thanthat of amino and is equal to or slightly weaker than p-nitrophenol,thus theoretically replacement of hydroxyl by p-nitrophenol cannot becarried out.

We found that only when adding HOBT into the reaction mixture as acatalyst, strictly controlling the temperature to the screenedtemperature and controlling the reaction time, a bound HOBT transitionstate that could easily be left was formed to thereby effectivelyexchange with hydroxyl of camptothecin and thus to produce less reactionimpurities. The highest yield we obtained is 53.5%.

2) Whether the reaction between AA-Asn-amino benzylalcohol-p-nitrophenol-carbonic acid diester and drug R₅ via amino couldbe successfully taken place fully depends on selection of R₅.

The steric hindrance of the amino group in R₅ and the substituent of R₅have crucial effects on the linking reaction. The linking reactionbetween an aliphatic amino and R₁-R₂-R₃-Asn-amino benzylalcohol-p-nitrophenol-carbonic acid diester could produce high yield(such as MMAE) at mild condition. However, for the aromatic amino, noreaction product is obtained because the nucleophilicity of the amino isreduced due to its lone paired electron and the conjugation of aromaticring. By high-throughput screening and severe reaction conditions, forexample the linking reaction between Nimustine and R₁-R₂-R₃-Asn-aminobenzyl alcohol-p-nitrophenol-carbonic acid diester, we finally foundthat a small amount of products (yield 20%) could be obtained when usingDMAP as a base and reacting at 80-85 □.

3) In the above compounds R₁ has different effects on linking of R₄.

Different R₁ groups have significant effect on the linking reactionconditions between R₁-R₂-R₃-Asn-amino benzylalcohol-p-nitrophenol-carbonic acid diester and R₄. For example, linkingreaction between 4-N—(N—(N—(N-(6-maleimidocaproyl)-L-alanyl)-L-alanyl)-L-Asn)-amino benzylalcohol-p-nitrophenol-carbonic acid diester and camptothecin did notproduce a product. Therefore, different reactants and experimentalconditions should be screened in order to obtain the product. Forexample, 4-N—(N—(N—(N-(2-(2-methoxyethoxy)acetyl-L-alanyl)-L-alanyl)-L-asparaginyl)-amino benzylalcohol-p-nitrophenol-carbonic acid diester was used to react withcamptothecin under a special temperature condition, thereby producing acorresponding product.

Example 9: Compounds Produced by Linking to the Cleavable LinkerSpecifically Activated in a Tumor Microenvironment and Cytoxicity ChangeThereof

When R₁ is 2-(2-Methoxyethoxy)Acetyl, R₂ is Thr, and R₃ is Ala, thecompounds comprising an R₄ indicated below linking to the cleavablelinker could be screened by using similar catalysts used in thereactions for producing S1˜S3: camptothecin (S7), 10-hydroxycamptothecin (S8), topotecan (S9), Floxuridine (S10),5′-Deoxy-5-Fluorouridine (S11), cytarabine (S12), fludarabine (S13),etoposide (S14), Capecitabine (S15), gemcitabine (S16), vincristine(S17) and Epothilone B (S18), paclitaxel (S13′), Docetaxel (B13).

When R₁ is 2-(2-Methoxyethoxy)Acetyl, R₂ is Thr, and R₃ is Ala,compounds (R₄) that could be successfully linked to the cleavable linkerwhich is specifically activated in a tumor microenvironment include:daunorubicin (S19), epirubicin (S20), fludarabine (S21), gemcitabine(S22), nimustine (S23), mitoxantrone (S24), methotrexate (S25),Cytarabine (S26), Melphalan (S27), Doxorubicin (S28), and Mitomycin(E13).

TABLE 2 The properties and MS data of the synthetic compounds Molecularion Synthetic Cytoxicity No peak of MS efficiency % reduced (Multiple)S1 916 67 56 S2 885 19 145 S3 880 21 78 S4 1079 42 345 S5 1513 54 125 S61238 82 35 S7 954 56 432 S8 969 34 144 S9 1026 53 256 S10 851 87 89 S11851 43 46 S12 848 46 35 S13 970 25 78 S14 1193 46 463 S15 964 45 235 S16868 32 124 S17 1397 78 355 S18 1126 34 233 S19 1102 23 253 S20 1118 4539 S21 940 54 352 S22 838 22 121 S23 863 67 234 S24 1019 86 235 S25 102943 644 S26 818 34 123 S27 879 57 79 S28 1405 46 232 S13′ 1433 34 356 B131604 43 234 E13 886 56 454

Toxicity detection method: a standard universal test program was used toperform the in vitro cytotoxicity test. 2500 HEK293 cells were culturedin a 96-well plate, and allowed to grow overnight. Cytotoxic compoundsand their corresponding conjugated compounds were added into each wellin different concentrations, cultivated with the cells at 37° C. for 72hours, and then treated with MTT reagents. OD changes were read. Theapproximate multiples of the toxicity change were obtained by comparingthe IC50 of the modified compounds to their corresponding cytotoxiccompounds.

Example 10

Different compounds linking by the cleavable linker which isspecifically activated in a tumor microenvironment have differentactivation efficiencies.

The structure-efficacy relationship between the linking group and thegroups of the linked compound determines the activation effect. At 37□,1 mg/ml of S1, S2, S3, S4, S5 and S6 were added into 10 μg/ml acidifiedasparagine endopeptidase solution or a homogenate from different tumortissues (30 μg/ml), respectively. Reduction of reactant and increase ofproduct were detected by HPLC, thereby comparing the activationefficiency of these compounds (the ratio between the amount of thecompound released by cleaving by asparagine endopeptidase and theinitial amount of the compound, higher activation efficiency indicatingstronger activation efficiency). It was found that S1, S2, S3, S4 and S5exhibited very high activation efficiency by the tumor tissue, while S6had relatively low activation efficiency by the tumor tissue (Table 2).Our experimental results show that R₁ in S3, which is(N-hydroxylamino)-1,8-octandioic acid-1-monoacyl, could target and bindto metalloprotease MMP2 which is highly expressed in tumor, and R₁ inS5, which is 6-maleimido caproyl, could target and bind to cathepsinwhich is highly expressed in tumor. Thus, they have higher activationefficiency.

TABLE 3 Activation Efficiencies (%) of S1, S2, S3, S4, S5 and S6 Cellsthat produce tumor S1 S2 S3 S4 S5 S6 asparagine endopeptidase / 88.487.5 84.8 84.3 83.3 24.5 Human fibrosarcoma HT-1080 78.3 75.6 94.9 78.498.4 24.3 Human breast cancer MDA-MB435 67.3 78.4 70.1 83.5 96.7 25.6Human ovarian cancer SK-OV-3 78.3 74.6 94.3 78.4 97.4 23.5 Human coloncancer HT-29 63.7 78.3 81.7 83.5 78.4 22.4 Human chronic leukemia K56246.6 63.7 93.2 64.5 73.5 28.4 Human pancreatic cancer Panc-1 78.4 68.491.6 67.3 97.4 17.3 Human non-small cell lung A549 68.7 68.3 80.7 64.596.7 27.4 cancer Human prostate cancer PC-3 78.5 75.4 98.3 78.3 97.313.2 Human liver cancer Hepg2 86.4 63.7 94.5 67.3 67.3 26.7 Human renalcancer OS-RC-2 84.5 53.6 67.4 78.5 98.3 20.4 Human heart / 1.2 0.5 1.62.8 3.5 5.3

Example 11

Different compounds linking by a cleavable linker which is specificallyactivated in the tumor microenvironment have different activationefficiencies.

The structure-efficacy relationship between the linking group and thegroups of the linked compound determines the activation effect. At 37□,1 mg/ml of S7-S27 were added into 10 μg/ml acidified asparagineendopeptidase solution, respectively. Reduction of reactant and increaseof product were detected by HPLC, thereby comparing the activationefficiency of these compounds. The results are shown in Table 3.

TABLE 4 Activation Efficiencies (%) of S7-S27 Compound S7 S8 S9 S10 S11S12 S13 S14 Activation 75.7 65.5 86.4 95.4 66.2 73.6 79.6 85.3Efficiencies (%) Compound S15 S16 S17 S18 S19 S20 S21 S22 Activation84.6 13.4 89.4 93.5 89.3 76.7 95.4 97.5 Efficiencies (%) Compound S23S24 S25 S26 S27 S28 Activation Efficiencies (%) 91.5 90.7 74.4 78.5 73.566.5

From Table 4, it can be found that different compounds have differentactivation efficiencies by asparagine endopeptidase. The activationefficiencies of most of S7-S27 are all higher than 60%. S6 and S16 showvery low activation efficiency, which is less than 30%. Asparagineendopeptidase activates at the linkage between asparaginyl and 4-aminobenzyl alcohol. After cleaving by activation, 4-amino benzyl alcohol(4-aminobenzyl-OC(O)—) can be freely released, thereby releasing thedrug, R₄—H. The active center of asparagine endopeptidase locates at thebottom of its globular depression. The cleavage site should be close tothe active center. Thus, it is very important to determine whether thereis a steric hindrance to the cleavage site produced by the linkedcompound and to change the polarity of the linking site. According tothe above results, it is supposed that the steric hindrances andpolarities of S6 and S16 may affect their activation, resulting thatthey have relatively low activation efficiencies while other compoundshave relatively high activation efficiencies.

The results show that the cleavable linker which is specificallyactivated in a tumor microenvironment can link to and activate differentcompounds, in which the compounds may be classified into activatablecompounds and un-activatable compounds based on their different sterichindrance.

Example 12: Study on Efficacy of S1, S2, S3, S4, S5, S6, S16, S22 andS28 Injections in Nude Mice

Test purpose: to investigate the anti-tumor efficacy of S1, S2, S3, S4,S5, S6, S16, S22 and S28 via mouse tumor treatment model.

Test drug: S1, S2, S3, S4, S5, S6, S16, S22 and S28 injections, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Human breast cancer MDA-MB231 cells were purchased from American typeculture collection (ATCC) and identified according the specificationprovided by ATCC. Cells were cultivated in Dulbecco's minimum essentialmedium (DMEM culture medium) containing 10% fetal bovine serum at 37° C.and 5% CO₂. The cells were passaged for every three days and cellswithin the 15th passage were used.

2) Production of tumor. 5×10⁶ MDA-MB231 cells were subcutaneouslyinjected to the back of the nude mice. Mice were randomly grouped afterthe tumor reached at least 100 mm³. Then treatment began and the day onwhich the treatment began was day 1.

3) Course of treatment. According to the clinical application of S1, S2,S3, S4, S5, S6, S16, S22 and S28, drugs were intravenously injected(IV). A dose of 13.2 μmol/kg was used for S1, S2, S3, S4, S5, S6, S16,S22 and S28, camptothecin, capecitabine and daunorubicin, respectively.The drugs were administered once weekly for four weeks.

4) Grouping and test results are shown in Table 5.

TABLE 5 Effect of S1, S2, S3, S4, S5, S6, S16, S22 and S28 on treatmentof tumor on nude mice Inhibitory rate Number of Size of Tumor (mm³) ontumor (%) Group animal Day 10 Day 24 Day 10 Day 24 S1 group 10  56.53 ±14.36 124.44 ± 49.85  81 88 camptothecin 10 258.45 ± 57.43 847.46 ±157.56 15 19 S2 group 10  59.35 ± 35.53 89.53 ± 65.45 81 91 S3 group 10 85.67 ± 36.42 0 72 100 capecitabine 10 225.53 ± 74.45 946.43 ± 275.8626 9 S4 group 10  95.56 ± 57.54 64.68 ± 43.56 69 94 S5 group 10  63.67 ±46.64 46.45 ± 19.43 79 95.5 daunorubicin 10 174.78 ± 78.43 864.01 ±67.45  43 17 S6 group 10 235.5 ± 56.3   568.43 ± 245.56.67 23 45.3 S16group 10 246.76 ± 45.56 840.64 ± 345.6  23 19.21 S22 group 10 0 0 100100 S28 group 10 0 0 100 100 Control group 10 305.56 ± 75.75 1040.64 ±298.65  — — (physiological saline)

5) Results and discussion: as shown in Table 5, S1, S2, S3, S4, S5, S22and S28 exhibit strong inhibitory effect on tumor growth as compared tothe control group, the camptothecin group, the capecitabine group andthe daunorubicin group while S6 and S16 show less efficacy as they werenot activated. These indicate that the conjugates could significantlyimprove the efficacy of the drugs and the treatment effect is determinedby the cleaving efficiency. Comparing the structures of S6 with S4, S5and S19˜S27, it could be found that the amino of MMAE used for linkingis positioned at the two adjacent hydrophobic valines in MMAE. Afterlinking to 4-aminobenzyl-OC(O)—, the hydrophobic region of valine andthe little peptide of S6 are presented to a position which isunfavorable in relation to the active center of asparagineendopeptidase, resulting in formation of steric hindrance andobstruction of the active center of asparagine endopeptidase to approachthe cleavable bond. As a result, the efficiency of cleavage andactivation is very low. The activation efficiency of S6 is lower thanthe AANVV polypeptide. The 4-aminobenzyl-OC(O)— linker is no effect inthis compound. From the viewpoint of synthesis, it is more complicatedto add the 4-aminobenzyl-OC(O)— linker than to form a peptide bond. Onthe contrary, the amino position for linking in S4, S5 and S19-S27 isnot the amino group in their peptide, but it is the amino on thearomatic ring. We firstly discovered that the aromatic ring did notaffect the polarity of the linker. Thus, use of the 4-aminobenzyl-OC(O)—linker could eliminate the steric hindrance due to direct linking to anamino group and favorable activation results could be produced. Theactivation efficiency is generally higher than the compounds having adirect linking and this property is not limited to the compoundscontaining an aromatic ring.

Example 13: A Comparative Study on the Different Cleavable Linkers ofMMAE and Doxorubicin

When R₁ is 2-(2-Methoxyethoxy)Acetyl, and R₃ is Ala, activation studyand efficacy study were carried out by using compounds having differentcleavable linker. The study methods are identical to those in Examples10, 11 and 12. The in vitro activation efficiency was tested byasparagine endopeptidase and the tumor inhibition rate was tested byusing human breast cancer MDA-MB231 model.

Cytoxicity tumor synthesis Cleaving reduced inhibition No. linker forthe compounds efficiency efficiency (Multiples) rate (%)S6:PEG-TAN-PABC-MMAE) 2-(2-methoxyethoxy)acetyl- 84.5% 25.6% 35 45.4Thr-Ala-Asn-4-amino benzyl alcohol PEG-AAN-PABC-MMAE2-(2-methoxyethoxy)acetyl- 63.4% 14.4% 22 34.4 Ala-Ala-Asn-4-aminobenzyl alcohol AC-AAN-PABC-MMAE acetyl-Ala-Ala-Asn-4-amino 36.5% 3.4%15.5 16.7 benzyl alcohol CBZ-AAN-PABC-MMAE benzyloxy 24.6% 2.6% 8.3 9.7carbonyl-Ala-Ala-Asn-4-amino benzyl alcohol S28:PEG-AAN-PABC-DOX2-(2-methoxyethoxy)acetyl- 65.5% 99.5% 285.6 100 Ala-Ala-Asn-4-aminobenzyl alcohol PEG-AANL-DOX 2-(2-methoxyethoxy)acetyl- 45.5% 66.5% 110.260.3 AANL

Example 14

Compounds S29-S43 were synthesized by the same method as S1, except thatthe amino acids used as starting material were different. In thisExample, compounds having different amino acids were tested for theiractivation property and inhibitory rate on tumor. The test methods areidentical to the methods in Examples 4, 6, 8, 12 and 13. The testresults are shown in the following Table 7.

TABLE 7 The activation property and inhibitory rate on tumor forcompounds S28-S43 Inhibitory Inhibitory No. of Activation rate onActivation rate on Com- property tumor property tumor pound R₂ R₃ (%)(Day38) (%) (Day38) S29 Thr Thr 63.4%. 57.4% 63.4%. 57.4% S30 Thr Val46.3% 47.4% 46.3% 47.4% S31 Thr Asn 36.4% 46.5% 36.4% 46.5% S32 Val Ala68.4% 56.5% 68.4% 56.5% S33 Val Thr 34.5% 50.6% 34.5% 50.6% S34 Val Val54.3% 46.7% 54.3% 46.7% S35 Val Asn 34.5% 58.6% 34.5% 58.6% S36 Ile Ala35.5% 52.5% 35.5% 52.5% S37 Ile Thr 67.4% 46.7% 67.4% 46.7% S38 Ile Val38.5% 46.3% 38.5% 46.3% S39 Ile Asn 46.6% 48.4% 46.6% 48.4% S40 Ala Ala69.4% 80.5% 69.4% 80.5% S41 Ala Thr 78.3% 64.6% 78.3% 64.6% S42 Ala Val73.6% 66.6% 73.6% 66.6% S43 Ala Asn 65.4% 60.5% 65.4% 60.5%

Results and discussion: As shown in Table 7, S29-S43 exhibit a certainactivation property and inhibitory effect on tumor growth andmetastasis. The results also demonstrate that in the compounds beinghighly activated, R₂ can be any of Thr, Val, Ile and Ala, and R₃ can beany of Ala, Thr, Val and Asn.

Example 15: Study on Efficacy of 51, S2, S3, S4, S5 and S6 in D121 TumorImmune Model

Test purpose: to investigate the anti-tumor efficacy of S1, S2, S3, S4,S5 and S6 in a D121 lung cancer model for immune treatment.

Test drug: S1, S2, S3, S4, S5, S6, camptothecin, capecitabine anddaunorubicin, all used in 13.2 μmol/kg; PDL1 antibody, 5 μg/kg.

Animal: C57 mice of 6-8 weeks old, all female.

Production of Tumor Model:

1) D121 lung tumor cells were purchased from ATCC. Cells were cultivatedin DMEM culture solution containing 10% fetal bovine serum at 37° C. and5% CO₂. The cells were passaged for every three days and cells withinthe 15th passage were used.

2) Tumor immunization. 5×10⁵ D121 lung cancer cells (purchased fromATCC) which were killed by irradiation were intraperitoneally injectedto mice. The mice were injected for 3 times, once every two weeks. Afterimmunization, mice were injected with tumor cells and the drugs wereadministered weekly for 4 weeks.

3) Production of tumor. At day 32, 10⁶ live lung tumor cells weresubcutaneously injected to the back of the C57 mice immunized by tumor.Treatment began when the tumor grew to 0.3-0.4 cm.

4) Analysis on tumor CD8+ T cells. The tumor tissue was homogenated andindividual cells in the tumor were filtered, separated and washed bybuffer twice, then cultivated with the leucocyte common antigen CD45-PEand CD8-FITC marked antibodies for 1 hour at ambient temperature. Thecells were washed by phosphate buffer containing 1% fetal bovine serumtwice and then analyzed for the ratio of the T lymphocyte antigen (CD8)positive cells in the leucocyte common antigen (CD45) positive cells byflow cytometry.

5) Grouping and test results are shown in Table 8.

TABLE 8 Effect on inhibition of tumor and immune activation of S1, S2,S3, S4, S5, S6 and control Size of tumor Inhibitory rate on Number of(mm³) tumor CD8: D45 Group animal Day 18 Day 18 (%) Immune group,without D121 dead 8 1525.67 ± 314.6  — 6.8 tumor cells Immune group(Control group) 8 1357.57 ± 275.78  13.5 Immune group + S1 8 356.56 ±74.78  73.74 17.4 Immune group + camptothecin 8 889.56 ± 148.56 34.4713.2 Immune group + S2 8 379.67 ± 214.45 72.03 17.7 Immune group + S3 8425.67 ± 126.67 68.64 18.4 Immune group + capecitabine 8 953.65 ± 245.4329.75 13.6 Immune group + S4 8 316.78 ± 109.98 76.67 16.8 Immune group +S5 8 379.75 ± 125.64 72.03 17.4 Immune group + daunorubicin 8 1063.86 ±317.56  21.63 13.2 Immune group + S6 8 957.46 ± 257.87 29.47 13.0 Immunegroup + S1 + PDL1-antibody 8 81.78 ± 51.98 93.98 21.4 Immune group + 8816.64 ± 268.56 39.85 14.4 camptothecin + PDL1-antibody

6) Results and discussion. Treatment effects of S1, S2, S3, S4 and S5 onC57 mice were greatly improved as compared to the control group and theother treatment groups. The S6 group also has an improved treatmenteffect as compared to the daunorubicin group. S1 and PDL1-antibody showan excellent synergistic effect in promoting immunization and treatment.The results show that S1, S2, S3, S4 and S5 can inhibit tumor growth viaimproving immunization.

Example 16: Synthesis of Paclitaxel which is Specifically Activated inTumor Microenvironment 1) Synthesis of(R)-2-(2-(R)-(benzyloxycarbonyl)amino) propionylamino)methyl propionate(I)

N-benzyloxycarbonyl-L Ala (100 g, 0.45 mol) were dissolved inN,N-dimethylformamide (3 L). 1-hydroxylbenzotriazole (72.6 g, 0.54 mol)and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (103.3 g,0.54 mol) were added when stirring. After reacting for 1 hour understirring, the mixture was cooled in an ice bath and L-Ala methyl ester(46.2 g, 0.45 mol) and N,N-diisopropylethylamine (173.8 g, 1.34 mol) inthe N,N-dimethylformamide solution (1 L) was dropped into the mixture.After dropping, the mixture was stirred under ambient temperature (25□)for 10 hours. The solvents were removed by evaporation under reducedpressure. The crude product was dissolved in dichloromethane (2 L) andwashed subsequently by saturated ammonium chloride solution, water andsaturated sodium chloride solution. The organic phase was dried byanhydrous sodium sulphate. After removing the solvents by evaporationunder reduced pressure, the crude product was recrystallized to obtain awhite solid I (101 g, Yield 73.1%). LC-MS: 309[M+1]+.

2) Synthesis of (R)-2-(2-(R)-(benzyloxycarbonyl) amino)propionylamino)propionic acid (II)

(R)-2-(2-(R)-(benzyloxycarbonyl)amino) propionylamino)methyl propionate(100 g, 0.34 mol) were dissolved in a mixed solution of tetrahydrofuran(2 L) and water (1 L). The mixture was cooled to 0□ and 1M lithiumhydroxide solution (400 mL) were dropped into the mixture. The resultantmixture was stirred for reaction under ambient temperature (25□) for 10hours. Concentrated hydrochloric acid was dropped to adjust the pH to beless than 6. Most of tetrahydrofuran were removed by rotary evaporation.The residual water phase was extracted by dichloromethane (1 L×3). Theorganic phase was dried by anhydrous sodium sulphate. A white solid IIwas obtained after vaporizing and drying under reduced pressure (88 g;Yield, 92.2%). LC-MS: 295 [M+1]⁺.

3) Synthesis of (R)-2-((9H-fluorene-9-yl)methoxycarbonylamino)-4-(triphenylmethylamino)-1-hydroxymethylphenylsuccinic acid amide (III)

(R)-2-((9H-fluorene-9-yl) methoxycarbonylamino)-4-(triphenylmethylamino)butyrate (20 g, 0.03 mol),2-(7-azabenzotriazol)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(HATU) (15 g, 0.04 mol), N,N-dimethylformamide (200 mL) were added intoa 500 mL three-neck flask and stirred for 30 minutes. A solution of4-amino benzyl alcohol (4.1 g, 0.03 mol) in N,N-dimethylformamide (5mL), and N,N-diisopropyl ethylamine (8.7 g, 0.06 mol) were addedseparately under 0 □ and the mixture was stirred at ambient temperature(25□) for 3 hours. Most N,N-dimethylformamide were removed by rotaryevaporation. The residue was dissolved in ethyl acetate (200 mL), washedsubsequently by saturated ammonium chloride solution and saturatedsodium chloride solution and dried by anhydrous sodium sulphate. Afterfiltration, the solvent was removed by evaporation. The resultant crudeproduct was pulping by n-hexane/ethyl acetate (5/1,300 mL) to obtain awhite solid III (21.3 g, Yield 90%). LC-MS: 702 [M+1]⁺.

4) Synthesis of(R)-2-amino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinic acidamide (IV)

(R)-2-((9H-fluorene-9-yl)methoxycarbonylamino)-4-(triphenylmethylamino)-1-hydroxymethylphenylsuccinic acid amide (13.0 g, 18 mmol) were dissolved inN,N-dimethylformamide (80 mL). Piperidine (30 mL) was added and thenstirred at ambient temperature (25□) for 2 hours. The solvents wereremoved by evaporation under reduced pressure. And the resultant productwas dried under high vacuum within a vacuum drying oven (100□) to removea small quantity of piperidine. A pale yellow solid IV (8.43 g, yield:95%) was obtained, which could be used in the next step withoutpurification.

5) Synthesis of (R)-2-((R)-2-((R)-2-carboxybenzylamino) propionylamino)propionylamino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinicacid amide (V)

(R)-2-((R)-2-(carboxybenzylamino) propionylamino) propionic acid (6.0 g,20.4 mmol), benzotriazol-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 11.6 g, 30.6 mmol) and N,N-dimethylformamide(50 mL) were added into a three-neck flask and stirred for 30 minutes inan ice bath. A solution of(R)-2-amino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinic acidamide in N,N-dimethylformamide (50 mL), and N,N-diisopropylethylamine(7.89 g, 61.2 mmol) were added separately under 0° C. The resultantmixture was stirred for 17 hours at ambient temperature (25° C.). Thesolvents were removed by evaporation under reduced pressure. The residuewas dissolved in acetyl acetate (200 mL), washed subsequently bysaturated ammonium chloride solution (100 mL) and saturated sodiumchloride solution (100 mL) and dried by anhydrous sodium sulphate. Afterfiltration, the solvent was removed by evaporation. The resultant crudeproduct was recrystallized to obtain a white solid V (15 g, Yield 97%).LC-MS: 756 [M+1]⁺.

6) Synthesis of (R)-2-((R)-2-((R)-aminopropionylamino)propionylamino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinicacid amide (VI)

(R)-2-((R)-2-((R)-2-carboxybenzylamino) propionylamino)propionylamino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinicacid amide (5.0 g, 6.61 mmol) were dissolved in THF (150 mL). 10% Pd/C(1 g) was added. After introducing hydrogen gas, the resultant mixturewas stirred for reaction under normal temperature (22° C.) for 5 hours.Pd/C was removed by filtration and washed by methanol (100 mL). Thefiltrates and the washing solutions were pooled. Most solvents wereremoved by rotary evaporation to obtain a crude product. After silicagel column chromatography (200-300 mesh,dichloromethane/methanol=20/1-10/1, 2.5 L), a white solid VI wasobtained (2.0 g, Yield 49%). LC-MS: 622 [M+1]⁺.

7) Synthesis of (R)-2-((R)-2-((R)-2-(methoxyethoxyacetylamino)propionylamino)propionylamino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinicacid amide (VII)

2-(2-methoxyethoxy) acetic acid (432 mg, 3.22 mmol) were dissolved inN,N-dimethylformamide (20 mL). Benzotriazol-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (1.83 g, 4.83 mmol) were added and stirred for 30minutes. Then (R)-2-((R)-2-((R)-aminopropionylamino)propionylamino(triphenylmethylamino)-1-hydroxymethylphenyl succinic acidamide (2.0 g, 3.22 mmol) and N,N-diisopropylethylamine (1.24 g, 9.61mmol) in N,N-dimethylformamide (20 mL) were dropped into the resultantmixture. After dropping, the temperature was slowly raised to ambienttemperature (25° C.) and then the mixture was stirred for 10 hours. Mostof N,N-dimethylformamide were removed by evaporation under reducedpressure. The residue was dissolved in acetyl acetate (200 mL), washedsubsequently by saturated ammonium chloride solution (150 mL) andsaturated sodium chloride solution (150 mL) and dried by anhydroussodium sulphate. After filtration, the solvent was removed by rotaryevaporation. The resultant crude product was purified by silica gelcolumn chromatography (200-300 mesh, dichloromethane/methanol=20/1-10/1,2 L) to obtain a white solid VII (1.2 g, Yield 50%). LC-MS: 738 [M+1]⁺.

8) Synthesis of (R)-2-((R)-2-((R)-2-(methoxyethoxyacetylamino)propionylamino) propionylamino-1-hydroxymethylphenyl succinic aciddiamide (VIII)

(R)-2-((R)-2-((R)-2-(methoxyethoxyacetylamino) propionylamino)propionylamino-4-(triphenylmethylamino)-1-hydroxymethylphenyl succinicacid amide (VII) (1.0 g, 1.36 mmol) were dissolved in dichloromethane(10 mL). Trifluoroacetic acid (2 mL) were added and then the resultantmixture was stirred at ambient temperature (25° C.) for 5 hours. Thereaction solution was washed by water (20 mL) and separated. The organicphase was dried by anhydrous sodium sulphate and the solvents wereremoved by evaporation under reduced pressure. The residualtrifluoroacetic acid was removed by evaporation. The resultant crudeproduct was purified by silica gel column chromatography (200-300 mesh,dichloromethane/methanol=15/1-8/1, 1.5 L) to obtain VIII (600 mg, Yield89%). LC-MS: 496 [M+1]⁺.

9) Synthesis of 4-((R)-2-((R)-2-((R)-2-(methoxyethoxyacetylamino)propionylamino) propionylamino-4-aminocarboxybutyryl))aminobenzylp-nitrophenylcarbonate ester (IX)

(R)-2-((R)-2-((R)-2-(methoxyethoxyacetylamino) propionylamino)propionylamino-1-hydroxymethylphenyl Succinimide (500 mg, 1.01 mmol)were added into a 50 mL three-neck flask, dissolved in dichloromethane(10 mL). The resultant mixture was cooled to 0-5° C. p-nitrophenylchloroformate (406 mg, 2.02 mmol) and pyridine (160 mg, 2.03 mmol) weresubsequently dropped into the mixture under protection by nitrogen gas.After dropping, the resultant mixture was stirred at ambient temperature(25° C.) for 18 hours. The reaction solution was washed by water (10 mL)and separated. The organic phase was dried by anhydrous sodium sulphateand the solvents were removed by rotary evaporation. The resultant crudeproduct was purified by silica gel column chromatography (200-300 mesh,dichloromethane/methanol=30/1-20/1, 1 L) to obtain IX (450 mg, Yield67%). LC-MS: 661 [M+1]⁺.

10) Synthesis of 2-(2-methoxyethoxy)acetylamino-L-Ala-L-Ala-L-Asn-p-amino-benzyl-paclitaxel (S1′)

4-((R)-2-((R)-2-((R)-2-(methoxyethoxyacetylamino)propionylamino)propionylamino-4-amino-succinyl))amino-benzyl-paclitaxel-carbonic aciddiester (250 mg, 0.293 mmol) and paclitaxel (194 mg, 0.293 mmol) weredissolved in anhydrous N,N-dimethylformamide (10 mL). The resultantmixture was cooled to 0□ and then 4-dimethyl pyridine (DMAP) (54 mg,0.44 mmol) were added. The resultant mixture was stirred at ambienttemperature (25□) for 18 hours. The reaction solution was poured intoacetyl acetate (20 mL), the organic phase was combined and washedsubsequently by water (30 mL) and dried by anhydrous sodium sulphate.The solvents were removed by rotary evaporation to obtain a crudeproduct. The crude product was purified by silica gel columnchromatography (200-300 mesh, dichloromethane/methanol=20/1-15/1, 500mL) to obtain the target product S1 (150 mg, Yield 57%). LC-MS: 1375[M+1]⁺. The LC-MS result showed that the corresponding mass-to-chargeratio of elution peak 8.59 was 1375, which are consistent to itscalculated mass-to-charge ratio of 1374.5.

S2′, S3′ and S4′ were synthesized by making reference to S1′, as shownin the below table, except that the acetic acids substituted by alkoxygroup used in step 7 have different molecular weights. When synthesizingS2′, 3, 6, 9, 12, 15, 18-hexaoxanonadecanoic acid was used to replace2-(2-methoxyethoxy) acetic acid; in synthesis of S3′, 3, 6, 9, 12, 15,18, 21, 24, 27, 30, 33, 36-dodecaoxaheptatriacontanoic acid was used toreplace 2-(2-methoxyethoxy) acetic acid; and in synthesis of S4′,purchased long chain polyoxa fatty acid (customized from GL Biochem(Shanghai) Ltd., n=300) was used to replace 2-(2-methoxyethoxy) aceticacid. According to mass spectrum (MS) detection results, themass-to-charge ratios of S2′ and S3′ are 1551 and 1816, respectively,which are consistent to their calculated molecular weights, 1550.6 and1815.9. According to Matrix-Assisted Laser Desorption/Ionization Time ofFlight Mass Spectrometry (MALDI-TOF-MS), S4's molecular weight is about14524, which is consistent with its calculated molecular weight,14524.7.

TABLE 9 No. n Character Molecular weight by MS Fluorescence Output(milligram) Yield S1′ 1 White powder 1375 None 150 57% S2′ 5 Whitepowder 1551 None 178 48% S3′ 11 White powder 1816 None 159 56% S4′ 150White powder 14524 None 525 38%

Example 17: Synthesis of S10′-524′

The synthetic method was similar to that for S1′, except for thestarting amino acids used for linking are different, as shown in Table10. Corresponding R₂ amino acid and R₃ amino acid were dissolved inN,N-dimethylformamide, respectively. The same condensating agent,1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, was addedand reactions were allowed to take place at 0-25□ for 0.5-2 hours. ThenAsn was added and reaction was taken place at 0-25□ for 2-24 hours toobtain a tripeptide. As determined by mass spectrum (MS), the molecularweights of S10′-S24′ (n=1) are shown in the following table, which areconsistent with their calculated molecular weights.

TABLE 10 MS Molecular Output No. R₂ amino acid R₃ amino acid detectionweight Character (mg) Yield S10′ Ala Thr 1405 1405.03 white powder 9767% S11′ Ala Val 1403 1402.98 white powder 113 43% S12′ Ala Asn 14181417.95 white powder 135 25% S13′ Thr Ala 1405 1405.03 white powder 32178% S14′ Thr Thr 1435 1435.05 white powder 79 57% S15′ Thr Val 14331433.08 white powder 41 24% S16′ Thr Asn 1448 1448.05 white powder 13557% S17′ Val Ala 1403 1403.05 white powder 312 68% S18′ Val Thr 14331433.08 white powder 112 45% S19′ Val Val 1431 1431.11 white powder 6836% S20′ Val Asn 1446 1446.08 white powder 39 53% S21′ Ile Ala 14171417.08 white powder 18 19% S22′ Ile Thr 1447 1447.11 white powder 2732% S23′ Ile Val 1445 1445.14 white powder 74 34% S24′ Ile Asn 14601460.11 white powder 47 51%

Example 18: Solubility Comparison of Present Water-Soluble Paclitaxelfor Targeted Activation in Tumor Microenvironment and Control Compoundson the Formulation of the Drug

(1) Sample Treatment

Compounds S1′, S2′, S3′ and S4′(prepared in example 16) and variouscontrol compounds C1, C2, C3, C4, C5 and C6 were lyophilized (−70□),separately packing in a sterile room. Before animal test, S1′, S2′, S3′and S4′ were dissolved by solvent 1 (injectable water) or solvent 2 (50%injectable water, 42%˜49% propanediol, 1%˜8% Tween80) in sterile room.S1′, S2′, S3′ and S4′ could completely dissolved in both solvent 1 andsolvent 2, achieving a concentration of 10 mg/ml, and can be diluted byinjectable water to the desired concentration. On the contrary,comparative compounds (C1, C2, C3, C4, C5) did not satisfy theformulating requirement, as shown in Table 11.

TABLE 11 Effect of absence of similar components in control compounds orlinkage to Paclitaxel at its 7- or 2-position (i.e., linking the groupto the OH at 7- or 2-position of Paclitaxel) on the solubility of thedrug Compound Solvent 1 Solvent 2 C1: AAN -group 2- Paclitaxel (linkingat 2-position) insoluble insoluble C2: group 1- AANL - Paclitaxelinsoluble insoluble (linking at 2-position) C3: AAN - Paclitaxel(linking at 2-position) insoluble insoluble C4: group 1- AAN -group 2-Paclitaxel insoluble insoluble (linking at 7-position) C5: group 1- AANL-group 2- Paclitaxel insoluble insoluble (linking at 2-position) C6:group 1- AANK -group 2- Paclitaxel soluble soluble (linking at2-position) S1′ soluble soluble S2′ soluble soluble S3′ soluble solubleS4′ soluble soluble

In Table 11, AAN, AANL and AANK indicate the linkage formed by smallpeptides in the compounds, A is Ala, N is Asn, L is Leu and K is Lys.Solvent 1 is injectable water, solvent 2 contains 50% injectable water,45%˜49% propanediol, 1%˜5% Tween80. The dissolution concentration is 10mg/ml.

According to Table 11, solubility of the present Paclitaxel derivativesis significantly changed, with increased solubility in solvent 1 or 2.Change in solubility may greatly affect the formulation scheme of adrug. Solubility of comparative compounds (C1, C2, C3, C4, C5) did notsatisfy the formulating requirement. As compared to the traditionalPaclitaxel which is insoluble in water, 51′, S2′, S3′ and S4′ can beused to produce a soluble formulation. Their injection doses andefficacies can be improved and auxiliary materials that cause allergygenerally used for Paclitaxel can be avoided, indicating that they havea promising innovation and prospect of use.

Example 19: Methods for Determining the Contents of S1′, S2′, S3′ andS4′ in Respective Products and their Content Ranges

As detected by analytic HPLC (Agilent 1220 series, C8 column 5 μm, 4.6mm ID×250 mm; the mobile phase is 0-95% acetonitrile (ACN)), thepurities of S1′, S2′, S3′ and S4′ are all in the range of 95-99%.

Example 20: Activation Efficiency of Present Paclitaxel Derivatives forTargeted Activation in Tumor Microenvironment

Solvent (50% injectable water, 45%-49% alcohol, 1%-5% Tween 80) was usedto dissolve sample compound S1′, S2′, S3′ and S4′, and they were dilutedfor ten times to a concentration of 1 mg/ml. At 37□, sample compoundswere added into 100 μg acidized tumor tissue homogenates (pH6.0) in aconcentration of 1 mg/ml. The enzyme in tumor tissue homogenates couldrelease Paclitaxel. Reduction of compounds and increase of Paclitaxelwere detected by HPLC, thereby comparing the activation efficiency ofthe drugs by the tumor tissue. It was found that the current compoundsS1′, S2′, S3′ and S4′ exhibited highest activation efficiency among thescreened compounds.

TABLE 12 Activation ratio (%) of S1′, S2′, S3′ and S4′ in homogenatesfrom different tumor tissues Activation ratio (%) in homogenates fromCells producing different tumor tissues Different tumor tissues tumorS1′ S2′ S3′ S4′ Human fibrosarcoma HT-1080 74.7 75.4 67.9 74.6 Humanbreast cancer MDA-MB435 92.3 91.4 90.4 92.8 Human ovarian cancer SK-OV-388.4 84.6 79.3 63.8 Human colon cancer HT-29 79.4 89.9 91.4 90.6 Humanchronic leukemia K562 64.7 73.3 70.2 74.2 Human pancreatic cancer Panc-194.8 93.8 91.5 93.1 Human non-small cell lung A549 86.4 89.4 81.4 83.6cancer Human prostate cancer PC-3 97.3 98.4 96.3 93.5 Human liver cancerHepg2 95.3 84.6 83.5 74.2 Human renal cancer OS-RC-2 86.4 91.5 86.4 90.5Human heart none none none none

Solvent (50% injectable water, 42%-49% alcohol, 1%-8% Tween 80) was usedto dissolve sample compounds S1′, S2′, S3′ and S4′, and they werediluted for ten times to a concentration of 1 mg/ml. At 37□, samplecompounds were added into 100 μg acidized human breast cancer(MDA-MB435) tumor tissue homogenates (pH6.0) in a concentration of 1mg/ml. The enzyme in tumor tissue homogenates could release Paclitaxel.Reduction of compounds and increase of Paclitaxel were detected by HPLC,thereby comparing the activation efficiency of the drugs by the tumortissue. Results were showed in table 13.

TABLE 13 Effect of absence of similar components in control compounds onactivation of the drugs activation efficiency Compounds (%) C1:AAN-group 2-Paclitaxel (linking at 2-position) 67.4 C2: group1-AAN-Paclitaxel (linking at 2-position) 54.8 C3: AAN-Paclitaxel(linking at 2-position) 34.9 C4: group 1-AAN-group 2-Paclitaxel (linkingat 7-position) 12.1 C5: group 1-AANL-group 2-Paclitaxel (linking at2-position) 57.4 C6: group 1-AANK-group 2-Paclitaxel (linking at2-position) 47.7 S1′ 94.3 S2′ 93.1 S3′ 91.5 S4′ 87.8

According to the results, different groups in the present Paclitaxel fortargeted activation in tumor microenvironment have various effects onthe activation of Paclitaxel drugs in tumor tissue. The mutualstructure-efficacy of Paclitaxel with the groups linked determined thetargeting and activation effects in tissues. Activation of S1′, S2′, S3′and S4′ in different tumor types (10 kinds) proved their broad treatmentspectrum (Table 13). Meanwhile, certain compounds produced in thescreening were compared, and the activation efficiency in the same humanbreast cancer MDA-MB435 tissue was examined. It was proved that therespective group selection in S1′, S2′, S3′ and S4′ had relativelyhigher activation efficiency (Table 13).

The Paclitaxel derivatives for targeted activation in tumormicroenvironment of the present disclosure were based on a great amountof synthetic experiments. In these experiments, we designed a lot ofcomplicated compounds having different linking manners. Then thecomplicated compounds were linked to position 2 or 7 of Paclitaxel, thatis, they were linked to Paclitaxel via the OH at position 2 or position7. The resultant Paclitaxel derivatives were screened through activationefficiency in tumor tissues. The screened derivatives were furtherscreened through inhibition of tumor for R₂, R₃ and n. The activatedsite that is specific to the tumor tissue locates between AAN and group2. After cleaving by activation, group 2 can be freely released, therebyreleasing Paclitaxel. Because the active center of asparagineendopeptidase locates at the bottom of its globular depression and thecleavage site should be close to the active center, it is very importantif there is a steric hindrance to the cleavage site produced by thecomplicated compounds.

According to the screening results, it is presumed that linking of group2 may effectively avoid steric hindrance produced by directly linkingPaclitaxel, which thereby not affecting approach of asparagineendopeptidase. And, the structure-efficacy of group 1 may increase thepolarity of the cleavage site, which allows the more water-solubleprotease to be easily to approach the cleavage site and thereby toincrease the cleaving efficiency. Linking to position 2 of Paclitaxelcould obviously reduce steric hindrance produced by Paclitaxel toprotease, expose more groups, each of which as a whole is hydrophilic,and increase cleaving efficiency and water solubility. Whereas anadditional polar amino acid K or L would decrease the activationefficiency.

Example 21: Detection of Maximum Tolerated Dose (MTD) by IntravenousInjection of the Paclitaxel Derivatives for Targeted Activation in TumorMicroenvironment

Test purpose: to investigate the acute toxicity of the presentPaclitaxel derivatives via detecting MTD (maximum tolerated dose) byintravenous injection.

Test drugs: Solvent (50% injectable water, 42%-49% alcohol, 1%-8% Tween80) was used to dissolve sample compounds S1′, S2′, S3′ and S4′, dilutedto corresponding concentrations by physiological saline when testing, toprepare S1′, S2′, S3′ and S4′ injections.

Animal: the first class BALB/C mice purchased from SHANGHAI SLACLABORATORY ANIMAL CO. LTD, weighing 19-21 g and all mice being female.

Method and results: 42 BALB/C mice were randomly divided into 7 groupsaccording to their body weights, with 6 mice in each group. As shown inTable 14, the mice were intravenously injected with S1′, S2′, S3′ andS4′ for just one time in a dose of 0 mg/kg, 25 mg/kg, 50 mg/kg, 60mg/kg, 70 mg/kg, 80 mg/kg, and 960 mg/kg. Control tests were performedby injecting 0.2 ml physiological saline or Paclitaxel (purchased fromYoucare Pharmaceutical Group Co., Ltd). Animals were observed for 17continuous days for presence or absence of the following behaviors oneach day: pilo-erection, hair tousle and lackluster, lethargy, stoop andirritable reaction, and body weight and death were recorded. Bloodsamples were taken on the 3, 5 and 14 days for counting the whole bloodcells. Animals were anatomized on day 14 to take the heart, liver,kidney, lung, spleen, and pancreas for HE staining.

TABLE 14 Comparison of mortality rates of test mice receiving differentdoses of S1′, S2′, S3′ and S4′ injections, physiological saline orPaclitaxel injection Number of Mortality Dose Number of dead rate Group(mg/kg) animal animal (%) 1 physiological  0 mg/kg 10 0 0 saline 2 S1′125 mg/kg 10 0 0 3 S1′ 150 mg/kg 10 0 0 4 S1′ 175 mg/kg 10 0 0 5 S1′ 200mg/kg 10 1 10 6 S2′ 125 mg/kg 10 0 0 7 S2′ 150 mg/kg 10 0 0 8 S2′ 175mg/kg 10 0 0 9 S2′ 200 mg/kg 10 1 10 10 S3′ 125 mg/kg 10 0 0 11 S3′ 150mg/kg 10 0 0 12 S3′ 175 mg/kg 10 0 0 13 S3′ 200 mg/kg 10 1 10 14 S4′ 125mg/kg 10 0 0 15 S4′ 150 mg/kg 10 0 0 16 S4′ 175 mg/kg 10 0 0 17 S4′ 200mg/kg 10 0 10 18 Paclitaxel  25 mg/kg 10 0 0 19 Paclitaxel  30 mg/kg 101 10% 20 Paclitaxel  35 mg/kg 10 4 40% 21 Paclitaxel  40 mg/kg 10 8 90%

Results and discussions: no pilo-erection, hair tousle and lackluster,lethargy, stoop, irritable reaction and death were observed in micereceiving 90 mg/kg S1′, S2′, S3′ and S4′ injections. As shown in Table11, the MTD of the S1′ and S2′ injections were about 90 mg/kg, which isfar beyond the MTD of Paclitaxel, 6 mg/kg. The MTD for intravenousadministration of a test drug is an important reference index for drugtoxicity. The results indicate that the toxicity of the Paclitaxelreleased by targeted activation is significantly reduced as comparedwith Paclitaxel.

Example 22: Study on Efficacy of S1′, S2′, S3′ and S4′ Injections inNude Mice

Test purpose: to investigate the anti-tumor efficacy of S1′, S2′, S3′and S4′ in mice model for tumor treatment.

Test drug: S1′, S2′, S3′ and S4′ injections (same as Example 21) andPaclitaxel injection (purchased from Youcare Pharmaceutical Group Co.,Ltd), diluted to corresponding concentrations by physiological salinewhen testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female (purchased fromSHANGHAI SLAC LABORATORY ANIMAL CO. LTD).

2. Production of tumor model

1) Human prostate cancer PC-3 cells were purchased from American typeculture collection (ATCC) and identified according the specificationprovided by ATCC. Cells were cultivated in DMEM culture solutioncontaining 10% fetal bovine serum at 37° C. and 5% CO₂. The cells werepassaged for every three days and cells within the 15th passage wereused.

2) Production of tumor. 5×10⁶Panc-1 cells were subcutaneously injectedto the back of the nude mice. Mice were randomly grouped after the tumorreached at least 100 mm³. Then treatment began and the day on which thetreatment began was day 1.

3) Course of treatment According to the clinical application of S1′,S2′, S3′ and S4′, drugs were intravenously injected (IV). S1′, S2′, S3′and S4′ were administered in a dose of less than ⅙ MTD, i.e., 24 mg/kg,and Paclitaxel was administered in a dose of ⅓ MTD, i.e., 8 mg/kg. Thecontrol group was administered by physiological saline. Drugs wereadministered once weekly for four weeks.

4) Grouping and test results are shown in Table 15.

TABLE 15 Effect of S1′, S2′, S3′ and S4′, Paclitaxel and control groupon tumor treatment in nude mice inhibitory Number of Size of tumor (mm³)rate on tumor Group animal Day 10 Day 24 Day 10 Day 24 S1′ group 1076.42 ± 14.96 84.62 ± 45.94 35.7% 66.1% S2′ group 10 60.17 ± 30.26 42.39± 62.24 36.4% 83.01%  S3′ group 10 75.60 ± 28.54 74.39 ± 48.94 49.4%70.2% S4′ group 10 73.35 ± 38.46 63.99 ± 47.13 42.9% 81.5% Paclitaxeltreatment 10 118.85 ± 36..47  249.54 ± 95.46   7.5% 27.9% group Controlgroup 10 268.12 ± 55.64    346.1 ± 104.74. / /

5) Results and discussions: As shown in Table 15, inhibition on tumorgrowth by S1′, S2′, S3′ and S4′ were greatly improved as compared withthe groups treating by Paclitaxel using the same molar concentration andthe control group.

Example 23: Study on Efficacy of S1′, S2′, S3′ and S4′ in D121 TumorImmune Model

Test purpose: to investigate the anti-tumor efficacy of S1′, S2′, S3′and S4′ in a D121 lung cancer model for immune treatment.

Animal: C57 mice of 6-8 weeks old, all female (purchased from SHANGHAISLAC LABORATORY ANIMAL CO. LTD).

Test drug: S1′, S2′, S3′ and S4′ injections (same as Example 21) andPaclitaxel injection (purchased from Youcare Pharmaceutical Group Co.,Ltd), diluted to corresponding concentrations by physiological salinewhen testing.

Production of Tumor Model:

1) D121 lung tumor cells were purchased from ATCC. Cells were cultivatedin DMEM culture solution containing 10% fetal bovine serum at 37° C. and5% CO₂. The cells were passaged for every three days and cells withinthe 15th passage were used.

2) Tumor immunization. 5×10⁵ D121 lung cancer cells (purchased fromATCC) which were killed by irradiation were intraperitoneally injectedto mice. The mice were injected for 3 times, once every two weeks. Afterimmunization, mice were injected with tumor cells and the drugs wereadministered weekly for 4 weeks. In table 16 below the immune group wasimmuned with D121 lung tumor cells and the group without dead D121 lungtumor cells was injected with physiological saline as controls.

3) Production of tumor. After immunization (4 weeks later), 10⁶ livelung tumor cells were subcutaneously injected to the back of the C57mice immunized by tumor. Treatment began when the tumor grew to 0.3-0.4cm. Tumor size (mm³) were noted and tumor inhibition rates werecalculated.

4) Analysis on tumor CD8+ T cells. The tumor tissue was homogenated andindividual cells in the tumor were filtered, separated and washed bybuffer twice, then cultivated with the leucocyte common antigen CD45-PEand CD8-FITC marked antibodies for 1 hour at ambient temperature. Thecells were washed by phosphate buffer containing 1% fetal bovine serumtwice and then analyzed for the ratio of the T lymphocyte antigen (CD8)positive cells in the leucocyte common antigen (CD45) positive cells byflow cytometry.

5) Grouping and test results are shown in Table 16.

TABLE 16 Effect on inhibition of tumor and immune activation of S1′,S2′, S3′ and S4′, Paclitaxel and control inhibitory rate Number of Sizeof tumor (mm³) on tumor % Group animal Day 18 Day 18 CD8: CD45 (%)Immune group, without D121 8 1887.56 ± 323.4  5.6 dead tumor cellsImmune group (Control group) 8 1574.46 ± 456.34  control 13.5 Immunegroup + S1′ 8 237.60 ± 156.42 84.9% 19.6 Immune group + S2′ 8 331.57 ±114.74 78.9% 18.1 Immune group + S3′ 8 357.63 ± 194.54 77.3% 16.7 Immunegroup + S4′ 8 304.55 ± 184.53 80.7% 17.8 Immune group + S1′ + PDL1 874.78 ± 27.25 95.3% 24.4 antibody Immune group + Paclitaxel 30 1210.28 ±375.46  23.1% 6.6 Immune group + Paclitaxel + 8 1334.90 ± 257.34  15.2%7.7 PDL1 antibody

6) Results and discussion. As shown in table 13, treatment effects ofS1′, S2′, S3′ and S4′ on C57 mice were greatly improved as compared tothe control group and the other treatment groups. S1′ and PDL1-antibodyshow an excellent synergistic effect in promoting immunization andtreatment. They can inhibit tumor growth via improving immunization.

Example 24: Study on Efficacy of S1′, S2′, S3′ and S4′ in BALB/C MiceModel for Tumor Metastasis

Test purpose: to investigate the anti-tumor efficacy of S1′, S2′, S3′and S4′ in BALB/C mice model for treatment of tumor metastasis.

Test drug: S1′, S2′, S3′ and S4′ injections (same as Example 31) andPaclitaxel injection (purchased from Youcare Pharmaceutical Group Co.,Ltd), diluted to corresponding concentrations by physiological salinewhen testing.

Method and Results:

1. Animal: the first class BALB/C mice of 6-8 weeks old, all female(purchased from SHANGHAI SLAC LABORATORY ANIMAL CO. LTD).

2. Production of tumor model

1) 4T1 cells were purchased from American type culture collection (ATCC)and identified according the specification provided by ATCC. Cells werecultivated in DMEM culture solution containing 10% fetal bovine serum at37° C. and 5% CO₂. The cells were passaged for every three days andcells within the 15th passage were used.

2) Production of tumor metastasis. 10⁶ T1 cells were subcutaneouslyinjected to the back of the BALB/C mice. Mice were randomly groupedafter the tumor grew to about 1.5 cm. The subcutaneous tumor was removedby surgery and drug treatment began. Mice were killed after anesthesiaon day 27. The whole lung was taken out and put into Bouin's solutionfor staining. The number of the tumor metastasized to lung was countedwith anatomical microscope.

3) Course of treatment

According to the clinical application of S1′, S2′, S3′ and S4′, drugswere intravenously injected (IV). S1′, S2′, S3′ and S4′ wereadministered in a dose of ⅙ MTD, i.e., 12 mg/kg, and Paclitaxel wasadministered in a dose of ⅙ MTD, i.e., 4 mg/kg. The control group wasadministered by physiological saline. Drugs were administered once forevery three days for 4 times.

4) Grouping and test results are shown in Table 17.

TABLE 17 Effects of S1′, S2′, S3′ and S4′, Paclitaxel and control oninhibition of tumor metastasis in BALB/C mice Number of InhibitoryNumber of metastasized rate on Group animal tumor metastasis S1′ Group10 2 ± 3 99.2% S2′ Group 10 8 ± 7 94.1% S3′ Group 10 13 ± 8  90.44%  S4′Group 10 15 ± 16 89.0% Paclitaxel 10 128 ± 25   5.9% treatment groupControl group 10 136.0 ± 46   /

5) Results and discussion. As shown in Table 17, the inhibitory effecton tumor metastasis of BALB/C mice was greatly improved afterintraperitoneal injection of S1′, S2′, S3′ and S4′, as compared with thePaclitaxel group and the control group, indicating that this kind ofdrugs exhibits an excellent efficacy on anti-tumor metastasis.

Example 25: Study on Efficacy of S1′ Injection in Multiple Tumor Models

Test purpose: to investigate the anti-tumor spectrum of S1′ throughmultiple tumor models from mice

Test drug: S1′ injection (same as Example 21), diluted to correspondingconcentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female (purchased fromSHANGHAI SLAC LABORATORY ANIMAL CO. LTD).

2. Production of tumor model

1) Corresponding tumor cells were purchased from American type culturecollection (ATCC) and identified according the specification provided byATCC. Cells were cultivated in DMEM culture solution containing 10%fetal bovine serum at 37° C. and 5% CO₂. The cells were passaged forevery three days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶ corresponding cells were subcutaneouslyinjected to the back of the nude mice. Mice were randomly grouped afterthe tumor reached at least 100 mm³. Then treatment began and the day onwhich the treatment began was day 1.

3) Course of treatment. According to the clinical application of S1′,S1′ was administered in a dose of ⅙ MTD, i.v., 17.6 μmol/kg. The controlgroup was administered by physiological saline. Animals wereadministered once weekly for three weeks.

4) Grouping and test results are shown in Table 18.

TABLE 18 Treatment effect of S1′ in multiple tumor models inhibitoryrate on Group Tumor cell tumor (Day 26) Human breast cancer MDA-MB43590.7% Human ovarian cancer SK-OV-3 85.6% Human colon cancer HT-29 89.7%Human chronic leukemia K562 77.9% Human colon caner HT1080 94.3% Humanpancreatic cancer Panc-1 88.59%  Human non-small cell lung cancer A54994.6% Human liver cancer Hepg2 84.3% Human renal cancer OS-RC-2 85.7%

5) Results and discussion. As shown in Table 18, S1′ shows an excellentefficacy in multiple tumor models, demonstrating that the anti-tumordrug has a wide anti-tumor spectrum.

Example 26: Activation Efficiency, Inhibitory Rate on Tumor andInhibitory Rate on Metastasis of S10′˜S24′

The activation efficiency, inhibitory rate on tumor and inhibitory rateon metastasis of S10′˜S24′ were examined respectively using methods sameas that in example 20, 22 and 24. Results were showed in table 19.

TABLE 19 activation efficiency, inhibitory rate on tumor and onmetastasis of S10′~S24′ inhibitory activation inhibitory rate onCompound efficiency rate on tumor metastasis No. R₂ R₃ (%) (%)(Day 38)(%) S10′ Ala Thr  65.4%. 65.6% 75.3% S11′ Ala Val 42.6% 46.2% 44.5% S12′Ala Asn 38.4% 49.5% 81.6% S13′ Thr Ala 75.7% 61.3% 87.4% S14′ Thr Thr37.5% 52.4% 29.4% S15′ Thr Val 54.6% 45.8% 39.3% S16′ Thr Asn 33.2%68.3% 56.8% S17′ Val Ala 30.6% 58.3% 64.8% S18′ Val Thr 65.8% 69.8%80.1% S19′ Val Val 38.5% 55.2% 68.3% S20′ Val Asn 43.5% 47.8% 71.4% S21′Ile Ala 49.6% 43.4% 63.9% S22′ Ile Thr 69.9% 59.5% 70.5% S23′ Ile Val57.5% 65.2% 45.5% S24′ Ile Asn   49% 47.48%  54.2%

In the present disclosure, other Paclitaxel derivatives for targetedactivation in tumor microenvironment were synthesized, of which n is anyinteger between 1-300, R₂ is Ala, Thr, Val or Ile; R₃ is Ala, Thr, Valor Asn. And they were subjected to activation test as done in Examples17, study on efficacy on tumor as done in Examples 23 and 24, study onefficacy of inhibiting metastasis as done in Example 25 and study onefficacy on multiple tumors as done in Example 26. Results showed thatthey had similar results to S1′-S4′. As demonstrated by the experiments,when n is in the range of 1-300, the inhibitory rate on tumor isslightly reduced as n increases. The activation activity also slightlydecreases and mass of drugs in the same mole increases, as n increases.However, the metabolic half life of the drug also increases as nincreases. Therefore, the entire efficacy is only slightly decreased andwhen n is in the range of 1-300, all compounds could produce similartechnical effect to S1′-S4′.

Example 27: Synthesis of Water-Soluble Paclitaxel for TargetedActivation 1). Synthesis of Di-(2-Methoxyethoxyacetyl)-L-Lysine EthylEster (I)

2-(2-methoxyethoxy) acetic acid (161 mg, 1.2 mmol) were dissolvedN,N-dimethylformamide (10 mL) and cooled in an ice bath.2-(7-azabenzotriazol)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(462 mg, 1.2 mmol), N,N-diisopropyl ethylamine (313 mg, 2.4 mmol) andL-lysine ethyl ester dihydrochloride (100 mg, 0.4 mmol) were added whenstirring. After addition, the resultant mixture was stirred at ambienttemperature overnight. The solvents were removed by evaporation underreduced pressure. The crude product was purified by reversed phasecolumn to obtain I (128 mg, Yield 77.8%).

2). Synthesis of di (2-methoxyethoxyacetyl)-L-lysine (II)

Di (2-methoxyethoxyacetyl)-L-lysine ethyl ester (I) (122 mg, 0.3 mmol)were dissolved in tetrahydrofuran (15 mL). An aqueous solution oflithium hydroxide (39 mg, 0.9 mmol) was dropped into the resultantmixture after it was cooled to 0° C. The resultant mixture was stirredat ambient temperature for 2 hours and then cooled in an ice bath. ThenpH was adjusted by concentrated hydrochloric acid to 2. Tetrahydrofuranwas removed by evaporation. The resultant product was freeze-dried toproduce a crude product II (112 mg, Yield 99%), which could be directlyused in the next step without purification.

3). Synthesis of di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn(Trt)-4-amino benzyl alcohol (III)

Di (2-methoxyethoxyacetyl)-L-lysine (112 mg, 0.3 mmol) were dissolved inN,N-dimethylformamide (10 mL). 3-(Diethoxyphosphoryloxy)-1, 2,3-benzotrizin-4-one (109 mg, 0.36 mmol), L-Ala-L-Ala-L-Asn (Trt)-4-aminobenzyl alcohol (188 mg, 0.3 mmol) and N,N-diisopropyl ethylamine (117mg, 0.9 mmol) were dropped into the resultant mixture after it wascooled to 0° C. After dropping, the resultant mixture was stirred atambient temperature overnight. The solvents were removed by evaporationunder reduced pressure. The crude product was purified by reversed phasecolumn to obtain III (159 mg, Yield 54.0%).

4). Synthesis of di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn(Trt)-4-aminobenzyl-4-nitrophenyl carbonate (IV)

Di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn (Trt)-4-amino benzylalcohol (167 mg, 0.17 mmol) dissolved in tetrahydrofuran (10 mL) wasadded into a three-neck flask. 4-nitrophenyl chloroformate (73 mg, 0.36mmol) and pyridine (39 mg, 0.50 mmol) were dropped into the resultantmixture after it was cooled to 0□. The resultant mixture was stirred atambient temperature overnight. The solvents were removed by evaporationunder reduced pressure. The crude product was purified by reversed phasecolumn to obtain IV (153 mg, Yield 78.5%).

5). Synthesis of di(2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenylcarbonate (V)

Di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn(Trt)-4-aminobenzyl-4-nitrophenyl carbonate (IV) (100 mg, 0.087 mmol)were dissolved in trifluoroacetic acid (1 mL). Two drops of water wereadded and then pumped by an oil pump immediately to obtain a crudeproduct V (80 mg), which could be directly used in the next step withoutpurification.

6). Synthesis of di(2-methoxyethoxyacetyl)-L-Ala-L-Ala-L-Asn-4-aminobenzyl-Paclitaxel (A1)

Di(2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenylcarbonate (80 mg, 0.088 mmol) and Paclitaxel (76 mg, 0.089 mmol) weredissolved by anhydrous N,N-dimethylformamide (10 mL) and cooled to 0□.DMAP (22 mg, 0.18 mmol) were added and then stirred at ambienttemperature overnight. Again Paclitaxel (38 mg, 0.044 mmol) was addedand the mixture was stirred overnight. The reaction solution was pouredinto ethyl acetate. The organic phases were pooled, washed by water,dried by anhydrous sodium sulphate. The solvents were removed by rotaryevaporation. The crude product was purified by reverse phase column toobtain the target product A1 (25 mg, Yield 37.5%). According to thedetection result by LC-MS, the mass-to-charge ratio of elution peak is1619, which is consistent with its calculated molecular weight.

7

A2, A3 and A4 were synthesized by making reference to A1, except thatthe acetic acids substituted by alkoxy group used in step 7 havedifferent molecular weights. When synthesizing A2, 3, 6, 9, 12, 15,18-hexaoxanonadecanoic acid was used to replace 2-(2-methoxyethoxy)acetic acid, in synthesis of A3, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36-dodecaoxaheptatriacontanoic acid was used to replace2-(2-methoxyethoxy) acetic acid, and in synthesis of A4, polyoxa fattyacid was used to replace 2-(2-methoxyethoxy) acetic acid. According tomass spectrum (MS) detection results, the mass-to-charge ratios of A2,A3 and A4 are 1619, 1972 and 2500, respectively, which are consistent totheir calculated molecular weights, 1619.71, 1972.13 and 2500.77.According to Matrix-Assisted Laser Desorption/Ionization Time of FlightMass Spectrometry (MALDI-TOF-MS), A4's molecular weight is about 14739,which is consistent with its calculated molecular weight, 14739.59, asshown in the table 20 below.

TABLE 20 Molecular weight by Output No. n Character MS Fluorescence(milligram) Yield A1 1 White powder 1619 None 25 37.5% A2 5 White powder1972 None 245 43.3% A3 11 White powder 2500 None 456 66.4% A4 150 Whitepowder 14739 None 645 34.6%

8

Compounds A10-A24 (n=5) were also prepared in the present disclosure bysimilar method for synthesizing A2, except that the starting amino acidsused for linking were different, as shown in Table 21. Corresponding R₂amino acid and R₃ amino acid were dissolved in N,N-dimethylformamide.The same condensating agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, was added respectively and reactions wereallowed to take place at 0-25□ for 0.5-2 hours. Then Asn was added andreaction was taken place at 0-25□ for 2-24 hours. The resultant waspurified to obtain a tripeptide. The tripeptide Ala-Ala-Asn was replacedto the synthesized intermediate to prepare A10-A24. Molecular weights ofA10-A24, as detected by mass spectrum (MS), are shown in Table 21, whichare consistent to their respective calculated molecular weights.

TABLE 21 No. of Molecular weight Calculated molecular Output Compound R₂R₃ by MS weight (milligram) Yield A10 Ala Thr 2002 2002.16 64 43% A11Ala Val 2000 2000.11 58 42% A12 Ala Asn 2015 2015.08 43 27% A13 Thr Ala2002 2002.16 48 38% A14 Thr Thr 2032 2032.18 45 37% A15 Thr Val 20302030.21 22 25% A16 Thr Asn 2045 2045.18 46 37% A17 Val Ala 2000 2000.1857 23% A18 Val Thr 2030 2030.21 43 35% A19 Val Val 2028 2028.24 23 23%A20 Val Asn 2043 2043.21 46 64% A21 Ile Ala 2014 2014.21 75 19% A22 IleThr 2044 2044.24 43 4% A23 Ile Val 2042 2042.27 23 33% A24 Ile Asn 20572057.24 66 45%

Example 28: Solubility Comparison of Present Water-Soluble Paclitaxelfor Targeted Activation and Control Compounds on the Formulation of theDrug

lyophilized (−70□) compounds A1, A2, A3, A4 and various controlcompounds C1, C2, C3, C4, C5 and C6 were separately packing in a sterileroom. Before animal test, A1, A2, A3 and A4 were dissolved by solvent 1(injectable water) or solvent 2 (45% alcohol, 55% injectable water) insterile room. A1, A2, A3 and A4 could completely dissolved in bothsolvent 1 and solvent 2, achieving a concentration of 10 mg/ml, and canbe diluted by injectable water to the desired concentration. On thecontrary, comparative compounds (C1, C2, C3, C4, C5 and C6) did notsatisfy the formulating requirement, as shown in Table 22.

TABLE 22 Effect of absence of similar components in control compounds orlinkage to Paclitaxel at its 7- or 2-position (i.e., linking the groupto the OH at 7- or 2-position of Paclitaxel) on the solubility of thedrug Compound Solvent 1 Solvent 2 C1: AAN -group 2- Paclitaxel (linkingat 2-position) insoluble insoluble C2: group 1- AANL - Paclitaxelinsoluble insoluble (linking at 2-position) C3: AAN - Paclitaxel(linking at 2-position) insoluble insoluble C4: group 1- AAN -group 2-Paclitaxel insoluble insoluble (linking at 7-position) C5: group 1- AANL-group 2- Paclitaxel insoluble insoluble (linking at 2-position) C6:group 1- AANK -group 2- Paclitaxel insoluble insoluble (linking at2-position) A1 insoluble soluble A2 soluble soluble A3 soluble solubleA4 soluble soluble

In Table 22, AAN, AANL and AANK indicate the linkage formed by smallpeptides in the compounds, A is Ala, N is Asn, L is Leu and K is Lys.

According to Table 22, Paclitaxel is insoluble in water, but itssolubility is significantly changed after modification, with increasedsolubility in water. Change in solubility may greatly affect theformulation scheme of a drug. As compared to the traditional Paclitaxelwhich is insoluble in water, A1, A2, A3 and A4 can be used to produce asoluble formulation. A1, A2, A3 and A4 can directly dissolve in water,Thus, their injection doses and efficacies can be improved and auxiliarymaterials that cause allergy generally used for Paclitaxel can beavoided. This is a great progress in drug development, and indicatesthat the water-soluble Paclitaxel for targeted activation in tumormicroenvironment has a promising innovation and prospect of use. On thecontrary, comparative compounds (C1, C2, C3, C4, C5 and C6) did notsatisfy the formulating requirement.

Example 29: Methods for Determining the Contents of A1, A2, A3 and A4 inRespective Products and their Content Ranges

As detected by analytic HPLC (Agilent 1220 series, C8 column 5 μm, 4.6mm ID×250 mm; the mobile phase is 0-95% acetonitrile (ACN)), thepurities of A1, A2, A3 and A4 are all in the range of 95-99%.

Example 30: Activation Efficiency of Present Water-Soluble PaclitaxelDerivatives for Targeted Activation in Tumor Microenvironment

Solvent (50% injectable water, 45%-49% alcohol, 1%-5% Tween 80) was usedto dissolve sample compound A1, A2, A3 and A4, and they were diluted forten times to a concentration of 1 mg/ml. At 370, sample compounds wereadded into 100 μg acidized tumor tissue homogenates (pH6.0) in aconcentration of 1 mg/ml. The enzyme in tumor tissue homogenates couldrelease Paclitaxel. Reduction of compounds and increase of Paclitaxelwere detected by HPLC, thereby comparing the activation efficiency ofthe drugs by the tumor tissue. It was found that the current compoundsA1, A2, A3 and A4 exhibited highest activation efficiency among thescreened compounds.

TABLE 23 Activation ratio (%) of A1, A2, A3 and A4 in homogenates fromdifferent tumor tissues Activation ratio (%) in homogenates from Cellsproducing different tumor tissues Different tumor tissues tumor A1 A2 A3A4 Human fibrosarcoma HT-1080 77.7 78.4 70.3 77.2 Human breast cancerMDA-MB435 95.6 94.4 93.4 97.8 Human ovarian cancer SK-OV-3 91.4 88.682.8 66.4 Human colon cancer HT-29 82.4 92.9 94.6 93.6 Human chronicleukemia K562 67.7 76.3 73.2 77.2 Human pancreatic cancer Panc-1 97.893.8 94.5 96.1 Human non-small cell A549 89.5 92.4 84.4 86.2 lung cancerHuman prostate cancer PC-3 100.3 101.4 99.3 96.5 Human liver cancerHepg2 98.3 87.6 86.5 77.0 Human renal cancer OS-RC-2 89.2 94.5 89.4 93.5Human heart none none none none

The affect to drug activation of similar ingredients in controlcompounds was evaluated. Solvent (50% injectable water, 42%-49% alcohol,1%-8% Tween 80) was used to dissolve sample compound A1, A2, A3 and A4,and they were diluted for ten times to a concentration of 1 mg/ml. At37□, sample compounds were added into 100 μg acidized human breastcancer (MDA-MB435) tumor tissue homogenates (pH6.0) in a concentrationof 1 mg/ml. The enzyme in tumor tissue homogenates could releasePaclitaxel. Reduction of compounds and increase of Paclitaxel weredetected by HPLC, thereby comparing the activation efficiency of thedrugs by the tumor tissue. Results were showed in table 24.

TABLE 24 activation efficiency Compounds (%) C1: AAN-group 2-Paclitaxel(linking at 2-position) 64.4 C2: group 1-AAN-Paclitaxel (linking at2-position) 51.9 C3: AAN-Paclitaxel (linking at 2-position) 32.7 C4:group 1-AAN-group 2-Paclitaxel (linking at 7-position) 11.6 C5: group1-AANL-group 2-Paclitaxel (linking at 2-position) 54.4 C6: group1-AANK-group 2-Paclitaxel (linking at 2-position) 43.3 A1 95.1 A2 96.3A3 94.5 A4 84.3

According to the results, different groups in the present Paclitaxel fortargeted activation in tumor microenvironment have various effects onthe activation of Paclitaxel drugs in tumor tissue. The mutualstructure-efficacy of Paclitaxel with the groups linked determined thetargeting and activation effects in tissues. Activation of A1, A2, A3and A4 in different tumor types (10 kinds) proved their broad treatmentspectrum (Table 24). Meanwhile, certain compounds produced in thescreening were compared, and the activation efficiency in the same humanbreast cancer MDA-MB435 tissue was examined. It was proved that therespective group selection in A1, A2, A3 and A4 had relatively higheractivation efficiency (Table 24).

The Paclitaxel derivatives (A1˜A4 and A10˜A23) for targeted activationin tumor microenvironment of the present disclosure were based on agreat amount of synthetic experiments. In these experiments, we designeda lot of complicated compounds having different linking manners. Thenthe complicated compounds were linked to position 2 or 7 of Paclitaxel,that is, they were linked to Paclitaxel via the OH at position 2 orposition 7. The resultant Paclitaxel derivatives were screened throughactivation efficiency in tumor tissues. The screened derivatives werefurther screened through inhibition of tumor for R₂, R₃ and n. Theactivated site that is specific to the tumor tissue locates between AANand group 2. After cleaving by activation, group 2 can be freelyreleased, thereby releasing Paclitaxel. Because the active center ofasparagine endopeptidase locates at the bottom of its globulardepression and the cleavage site should be close to the active center,it is very important if there is a steric hindrance to the cleavage siteproduced by the complicated compounds.

According to the screening results, it is presumed that linking of group2 may effectively avoid steric hindrance produced by directly linkingPaclitaxel, which thereby not affecting approach of asparagineendopeptidase. And, the structure-efficacy of group 1 may increase thepolarity of the cleavage site, which allows the more water-solubleprotease to be easily to approach the cleavage site and thereby toincrease the cleaving efficiency. Linking to position 2 of Paclitaxelcould obviously reduce steric hindrance produced by Paclitaxel toprotease, expose more groups, each of which as a whole is hydrophilic,and increase cleaving efficiency and water solubility. Whereas anadditional polar amino acid K or L would decrease the activationefficiency.

Example 31: Detection of Maximum Tolerated Dose (MTD) by IntravenousInjection of the Water-Soluble Paclitaxel Derivatives for TargetedActivation in Tumor Microenvironment

Test purpose: to investigate the acute toxicity of the presentPaclitaxel derivatives via detecting MTD by intravenous injection.

Test drugs: Solvent (50% injectable water, 42%-49% alcohol, 1%-8% Tween80) was used to dissolve sample compound A1, A2, A3 and A4, diluted tocorresponding concentrations by physiological saline when testing, toprepare A1, A2, A3 and A4 injections.

Animal: the first class BALB/C mice purchased from SHANGHAI SLACLABORATORY ANIMAL CO. LTD, weighing 19-21 g and all mice being female.

Method and results: 42 BALB/C mice were randomly divided into 7 groupsaccording to their body weights, with 6 mice in each group. As shown inTable 21, the mice were intravenously injected with A1, A2, A3 and A4for just one time in a dose of 0 mg/kg, 25 mg/kg, 50 mg/kg, 60 mg/kg, 70mg/kg, 80 mg/kg, and 960 mg/kg. Control tests were performed byinjecting 0.2 ml physiological saline or Paclitaxel (purchased fromYoucare Pharmaceutical Group Co., Ltd). Animals were observed for 17continuous days for presence or absence of the following behaviors oneach day: pilo-erection, hair tousle and lackluster, lethargy, stoop andirritable reaction, and body weight and death were recorded. Bloodsamples were taken on the 3, 5 and 14 days for counting the whole bloodcells. Animals were anatomized on day 14 to take the heart, liver,kidney, lung, spleen, and pancreas for HE staining.

TABLE 25 Comparison of mortality rates of test mice receiving differentdoses of A1, A2, A3 and A4 injections, physiological saline orPaclitaxel injection Dose Number of Number of Mortality Group (mg/kg)animal dead animal rate (%) 1 physiological  0 mg/kg 10 0 0 saline 2 A1125 mg/kg 10 0 0 3 A1 150 mg/kg 10 0 0 4 A1 175 mg/kg 10 0 0 5 A1 200mg/kg 10 0 0 6 A2 125 mg/kg 10 2 10 7 A2 150 mg/kg 10 0 0 8 A2 175 mg/kg10 0 0 9 A2 200 mg/kg 10 0 0 10 A3 125 mg/kg 10 1 10 11 A3 150 mg/kg 100 0 12 A3 175 mg/kg 10 0 0 13 A3 200 mg/kg 10 0 0 14 A4 125 mg/kg 10 220 15 A4 150 mg/kg 10 0 0 16 A4 175 mg/kg 10 0 0 17 A4 200 mg/kg 10 0 018 Paclitaxel  25 mg/kg 10 0 10 19 Paclitaxel  30 mg/kg 10 0 0 20Paclitaxel  35 mg/kg 10 1 10% 21 Paclitaxel  40 mg/kg 10 4 40%

Results and discussions: no pilo-erection, hair tousle and lackluster,lethargy, stoop, irritable reaction and death were observed in micereceiving 90 mg/kg A1, A2, A3 and A4 injections. As shown in Table 25,the MTD of the A1 and A2 injections were about 90 mg/kg, which is farbeyond the MTD of Paclitaxel, 6 mg/kg. The MTD for intravenousadministration of a test drug is an important reference index for drugtoxicity. The results indicate that the toxicity of the Paclitaxelreleased by targeted activation is significantly reduced as comparedwith Paclitaxel.

Example 32: Study on Efficacy of A1, A2, A3 and A4 Injections in NudeMice

Test purpose: to investigate the anti-tumor efficacy of A1, A2, A3 andA4 in mice model for tumor treatment.

Test drug: A1, A2, A3 and A4 injections (same as Example 31) andPaclitaxel injection (purchased from Youcare Pharmaceutical Group Co.,Ltd), diluted to corresponding concentrations by physiological salinewhen testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Human prostate cancer PC-3 cells were purchased from American typeculture collection (ATCC) and identified according the specificationprovided by ATCC. Cells were cultivated in DMEM culture solutioncontaining 10% fetal bovine serum at 37° C. and 5% CO₂. The cells werepassaged for every three days and cells within the 15th passage wereused.

2) Production of tumor. 5×10⁶Panc-1 cells were subcutaneously injectedto the back of the nude mice. Mice were randomly grouped after the tumorreached at least 100 mm³. Then treatment began and the day on which thetreatment began was day 1.

3) Course of treatment

According to the clinical application of A1, A2, A3 and A4, drugs wereintravenously injected (IV). A1, A2, A3 and A4 were administered in adose of less than ⅙ MTD, i.e., 24 mg/kg, and Paclitaxel was administeredin a dose of ⅓ MTD, i.e., 8 mg/kg. The control group was administered byphysiological saline. Drugs were administered once weekly for fourweeks.

4) Grouping and test results are shown in Table 26.

TABLE 26 Effect of A1, A2, A3 and A4, Paclitaxel and control group ontumor treatment in nude mice inhibitory rate Number of Size of tumor(mm³) on tumor Group animal Day 10 Day 24 Day 10 Day 24 A1 group 10 85.16 ± 58.4 89.78 ± 63.7 71.1% 76.4% A2 group 10  55.19 ± 56.2 44.43 ±47.9 81.3% 88.3% A3 group 10  82.72 ± 69.4.4 81.83 ± 89.2 71.9% 78.5% A4group 10  80.69 ± 68.2.4 67.09 ± 72.4 72.6% 82.4% Paclitaxel treatmentgroup 10 123.04 ± 125.3 252.49 ± 248.5 58.3% 33.7% Control group 10294.93 ± 275.8 380.71 ± 362.7 / /

5) Results and discussions: As shown in Table 26, inhibition on tumorgrowth by A1, A2, A3 and A4 were greatly improved as compared with thegroups treating by Paclitaxel using the same molar concentration and thecontrol group.

Example 33: Study on Efficacy of A1, A2, A3 and A4 in D121 Tumor ImmuneModel

Test purpose: to investigate the anti-tumor efficacy of A1, A2, A3 andA4 in a D121 lung cancer model for immune treatment.

Animal: C57 mice of 6-8 weeks old, all female.

Test drug: A1, A2, A3 and A4 injections (same as Example 31) andPaclitaxel injection (purchased from Youcare Pharmaceutical Group Co.,Ltd), diluted to corresponding concentrations by physiological salinewhen testing.

Production of Tumor Model:

1) D121 lung tumor cells were purchased from ATCC. Cells were cultivatedin DMEM culture solution containing 10% fetal bovine serum at 37° C. and5% CO₂. The cells were passaged for every three days and cells withinthe 15th passage were used.

2) Tumor immunization. 5×10⁵ D121 lung cancer cells (purchased fromATCC) which were killed by irradiation were intraperitoneally injectedto mice. The mice were injected for 3 times, once every two weeks. Afterimmunization, mice were injected with tumor cells and the drugs wereadministered weekly for 4 weeks. In the table below the immune group wasimmuned with D121 lung tumor cells and the group without dead D121 lungtumor cells was injected with physiological saline as controls.

3) Production of tumor. After immunization (4 weeks later), 10⁶ livelung tumor cells were subcutaneously injected to the back of the C57mice immunized by tumor. Treatment began when the tumor grew to 0.3-0.4cm. Tumor size (mm³) were noted and tumor inhibition rates werecalculated.

4) Analysis on tumor CD8+ T cells. The tumor tissue was homogenated andindividual cells in the tumor were filtered, separated and washed bybuffer twice, then cultivated with the leucocyte common antigen CD45-PEand CD8-FITC marked antibodies for 1 hour at ambient temperature. Thecells were washed by phosphate buffer containing 1% fetal bovine serumtwice and then analyzed for the ratio of the T lymphocyte antigen (CD8)positive cells in the leucocyte common antigen (CD45) positive cells byflow cytometry.

5) Grouping and test results are shown in Table 27.

TABLE 27 Effect on inhibition of tumor and immune activation of A1, A2,A3 and A4, Paclitaxel and control inhibitory rate Number of Size oftumor (mm³) on tumor % Group animal Day 18 Day 18 CD8: CD45 (%) Immunegroup, without D121 8 2076.316 ± 457.8 6.7 dead tumor cells Immune group(Control group) 8 1687.906 ± 341.6 14.2 Immune group + A1 8  261.36 ±178.3 84.52 20.4 Immune group + A2 8  375.727 ± 247.3 77.74 19.4 Immunegroup + A3 8  360.393 ± 312.7 78.65 17.3 Immune group + A4 8  324.005 ±268.4 80.80 16.9 Immune group + A1 + PDL1 8  71.258 ± 113.9 95.78 23.4antibody Immune group + Paclitaxel 8 1342.308 ± 379.3.8 20.47 5.7 Immunegroup + Paclitaxel + 8  1468.39 ± 412.8 13.00 7.2 PDL1 antibody

6) Results and discussion. As shown in table 27, treatment effects ofA1, A2, A3 and A4 on C57 mice were greatly improved as compared to thecontrol group and the other treatment groups. A1 and PDL1-antibody showan excellent synergistic effect in promoting immunization and treatment.They can inhibit tumor growth via improving immunization.

Example 34: Study on Efficacy of A1, A2, A3 and A4 in BALB/C Mice Modelfor Tumor Metastasis

Test purpose: to investigate the anti-tumor efficacy of A1, A2, A3 andA4 in BALB/C mice model for treatment of tumor metastasis.

Test drug: A1, A2, A3 and A4 injections (same as Example 31) andPaclitaxel injection (purchased from Youcare Pharmaceutical Group Co.,Ltd), diluted to corresponding concentrations by physiological salinewhen testing.

Method and Results:

1. Animal: BALB/C mice of 6-8 weeks old, all female.

2. Production of tumor model

1) 4T1 cells were purchased from American type culture collection (ATCC)and identified according the specification provided by ATCC. Cells werecultivated in DMEM culture solution containing 10% fetal bovine serum at37° C. and 5% CO₂. The cells were passaged for every three days andcells within the 15th passage were used.

2) Production of tumor metastasis. 10⁶ T1 cells were subcutaneouslyinjected to the back of the BALB/C mice. Mice were randomly groupedafter the tumor grew to about 1.5 cm. The subcutaneous tumor was removedby surgery and drug treatment began. Mice were killed after anesthesiaon day 27. The whole lung was taken out and put into Bouin's solutionfor staining. The number of the tumor metastasized to lung was countedwith anatomical microscope.

3) Course of treatment

According to the clinical application of A1, A2, A3 and A4, drugs wereintravenously injected (IV). A1, A2, A3 and A4 were administered in adose of ⅙ MTD, i.e., 12 mg/kg, and Paclitaxel was administered in a doseof ⅙ MTD, i.e., 4 mg/kg. The control group was administered byphysiological saline. Drugs were administered once for every three daysfor 4 times.

4) Grouping and test results are shown in Table 28.

TABLE 28 Effects of A1, A2, A3 and A4, Paclitaxel and control oninhibition of tumor metastasis in BALB/C mice Number of Number ofmetastasized Inhibitory rate on Group animal tumor metastasis A1 Group10 3 ± 4 97.9% A2 Group 10 9 ± 5 93.9% A3 Group 10 16 ± 9  89.1% A4Group 10 12 ± 18 91.8% Paclitaxel 10 137 ± 32  6.8% treatment groupControl group 10 147.0 ± 46   /

5) Results and discussion. As shown in Table 24, the inhibitory effecton tumor metastasis of BALB/C mice was greatly improved afterintraperitoneal injection of A1, A2, A3 and A4, as compared with thePaclitaxel group and the control group, indicating that this kind ofdrugs exhibits an excellent efficacy on anti-tumor metastasis.

Example 35: Study on Efficacy of A1 Injection in Multiple Tumor Models

Test purpose: to investigate the anti-tumor spectrum of A1 throughmultiple tumor models from mice

Test drug: A1 injection (same as Example 31), diluted to correspondingconcentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Corresponding tumor cells were purchased from American type culturecollection (ATCC) and identified according the specification provided byATCC. Cells were cultivated in DMEM culture solution containing 10%fetal bovine serum at 37° C. and 5% CO₂. The cells were passaged forevery three days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶ corresponding cells were subcutaneouslyinjected to the back of the nude mice. Mice were randomly grouped afterthe tumor reached at least 100 mm³. Then treatment began and the day onwhich the treatment began was day 1.

3) Course of treatment. According to the clinical application of A1, A1was administered in a dose of ⅙ MTD, i.v., 17.6 μmol/kg. The controlgroup was administered by physiological saline. Animals wereadministered once weekly for three weeks.

4) Grouping and test results are shown in Table 29.

TABLE 29 Treatment effect of A1 in multiple tumor models inhibitory rateon Group Tumor cell tumor (Day 26) Human breast cancer MDA-MB435 91.2%Human ovarian cancer SK-OV-3 84.4% Human colon cancer HT-29 87.5% Humanchronic leukemia K562 76.9% Human colon caner HT1080 95.6% Humanpancreatic cancer Panc-1 89.2% Human non-small cell lung cancer A54995.3% Human liver cancer Hepg2 85.4% Human renal cancer OS-RC-2 86.6%

5) Results and discussion. As shown in Table 29, A1 shows an excellentefficacy in multiple tumor models, demonstrating that the anti-tumordrug has a wide anti-tumor spectrum.

In other examples (A10˜A24) of the present disclosure, activationefficiency, inhibitory rate on tumor and inhibitory rate on metastasisof the present water-soluble Paclitaxel derivatives for targetedactivation with different amino acid structures were examined usingmethods same as that in example 30, 32 and 34. Results were showed intable 30.

TABLE 30 activation efficiency, inhibitory rate on tumor and onmetastasis of A10-A24 inhibitory inhibitory rate rate on Compoundactivation on tumor metastasis No. R₂ R₃ efficiency (%) (%) (Day 38) (%)A10 Ala Thr 70.4 69.85 82.5 A11 Ala Val 46.86 50.82 48.95 A12 Ala Asn42.24 53.79 89.76 A13 Thr Ala 83.27 67.1 96.14 A14 Thr Thr 41.25 57.6432.34 A15 Thr Val 60.06 50.6 43.23 A16 Thr Asn 36.52 75.13 62.48 A17 ValAla 33.66 64.13 71.28 A18 Val Thr 72.38 76.78 88.11 A19 Val Val 42.3560.72 75.13 A20 Val Asn 47.85 52.58 78.54 A21 Ile Ala 54.56 47.74 70.29A22 Ile Thr 76.89 65.45 77.55 A23 Ile Val 63.25 71.72 50.05 A24 Ile Asn76.44 86.4 78.45

Results and discussion. As shown in Table 30, compounds A10˜A24 could beactivated and had some effects on inhibition of tumor growth and onmetastasis, indicating the screening of inventors could optimize theactivation and treatment of tumor. It should be understood that theabove descriptions of preferred Examples are not intended to limit thesubject invention. After reading the above details, it is apparent tothe skilled artisan that amino acids at position R₂ and R₃ of thepresent drugs or compounds can be changed or replaced.

In some examples of the invention, other water-soluble Paclitaxelderivatives for targeted activation in tumor microenvironment weresynthesized, of which n is any integer between 1-150, R₂ is Ala, Thr,Val or Ile; R₃ is Ala, Thr, Val or Asn. And they were subjected toactivation test as done in Examples 28, study on efficacy on tumor asdone in Examples 32 and 33, study on efficacy of inhibiting metastasisas done in Example 34 and study on efficacy on multiple tumors as donein Example 35. Results showed that they had similar results to A1-A4. Asdemonstrated by the experiments, when n is in the range of 1-300, theinhibitory rate on tumor is slightly reduced as n increases. Theactivation activity also slightly decreases and mass of drugs in thesame mole increases, as n increases. However, the metabolic half life ofthe drug also increases as n increases. Therefore, the entire efficacyis only slightly decreased and when n is in the range of 1-150, allcompounds could produce similar technical effect to A1-A4.

Example 36: Synthesis of Water-Soluble and Targeting Activated DocetaxelB1 1. Synthesis of di (2-methoxyethoxyacetyl)-L-lysine ethyl ester (I)

2-(2-methoxyethoxy) acetic acid (161 mg, 1.2 mmol) were dissolvedN,N-dimethylformamide (10 mL) and cooled in an ice bath.2-(7-azabenzotriazol)-N,N,N′,N′-tetramethyluronium hexafluorophosphate(462 mg, 1.2 mmol), N,N-diisopropyl ethylamine (313 mg, 2.4 mmol) andL-lysine ethyl ester dihydrochloride (100 mg, 0.4 mmol) were added whenstirring. After addition, the resultant mixture was stirred at ambienttemperature overnight. The solvents were removed by evaporation underreduced pressure. The crude product was purified by reversed phasecolumn to obtain I (128 mg, Yield 77.8%).

2. Synthesis of di (2-methoxyethoxyacetyl)-L-lysine (II)

Di (2-methoxyethoxyacetyl)-L-lysine ethyl ester (I) (122 mg, 0.3 mmol)were dissolved in tetrahydrofuran (15 mL). An aqueous solution oflithium hydroxide (39 mg, 0.9 mmol) was dropped into the resultantmixture after it was cooled to 0° C. The resultant mixture was stirredat ambient temperature for 2 hours and then cooled in an ice bath. ThenpH was adjusted by concentrated hydrochloric acid to 2. Tetrahydrofuranwas removed by evaporation. The resultant product was freeze-dried toproduce a crude product II (112 mg, Yield 99%), which could be directlyused in the next step without purification.

3. Synthesis of di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn(Trt)-4-amino benzyl alcohol (III)

Di (2-methoxyethoxyacetyl)-L-lysine (112 mg, 0.3 mmol) were dissolved inN,N-dimethylformamide (10 mL). 3-(Diethoxyphosphoryloxy)-1, 2,3-benzotrizin-4-one (109 mg, 0.36 mmol), L-Ala-L-Ala-L-Asn (Trt)-4-aminobenzyl alcohol (188 mg, 0.3 mmol) and N,N-diisopropyl ethylamine (117mg, 0.9 mmol) were dropped into the resultant mixture after it wascooled to 0□. After dropping, the resultant mixture was stirred atambient temperature overnight. The solvents were removed by evaporationunder reduced pressure. The crude product was purified by reversed phasecolumn to obtain III (159 mg, Yield 54.0%).

4. Synthesis of di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn(Trt)-4-aminobenzyl-4-nitrophenyl carbonate (IV)

Di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn (Trt)-4-amino benzylalcohol (167 mg, 0.17 mmol) dissolved in tetrahydrofuran (10 mL) wasadded into a three-neck flask. 4-nitrophenyl chloroformate (73 mg, 0.36mmol) and pyridine (39 mg, 0.50 mmol) were dropped into the resultantmixture after it was cooled to 0□. The resultant mixture was stirred atambient temperature overnight. The solvents were removed by evaporationunder reduced pressure. The crude product was purified by reversed phasecolumn to obtain IV (153 mg, Yield 78.5%).

5. Synthesis of di(2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenylcarbonate (V)

Di (2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn(Trt)-4-aminobenzyl-4-nitrophenyl carbonate (IV) (100 mg, 0.087 mmol)were dissolved in trifluoroacetic acid (1 mL). Two drops of water wereadded and then pumped by an oil pump immediately to obtain a crudeproduct V (80 mg), which could be directly used in the next step withoutpurification.

6. Synthesis of di(2-methoxyethoxyacetyl)-L-Ala-L-Ala-L-Asn-4-aminobenzyl-Docetaxel (B1)

Di(2-methoxyethoxyacetyl)-L-Lys-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenylcarbonate (1176 mg, 1.3 mmol) and Docetaxel (1293 mg, 1.6 mmol) weredissolved by anhydrous N,N-dimethylformamide (20 mL) and cooled to 0□.DMAP (318 mg, 2.6 mmol) were added and then stirred at ambienttemperature overnight. The reaction solution was poured intodichloromethane. The organic phases were pooled, washed by water, driedby anhydrous sodium sulphate. The solvents were removed by rotaryevaporation. The crude product was purified by reverse phase column toobtain the target product B1 (511 mg, Yield 25%). According to thedetection result by mass spectrum (MS), the mass-to-charge ratio of B1is 1573, which is consistent with its calculated molecular weight,1573.69.

B2, B3 and B4 were synthesized by making reference to B1, except thatthe acetic acids substituted by alkoxy group used in step 1 havedifferent molecular weights. When synthesizing B2, 3, 6, 9, 12, 15,18-hexaoxanonadecanoic acid was used to replace 2-(2-methoxyethoxy)acetic acid, in synthesis of B3, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36-dodecaoxaheptatriacontanoic acid was used to replace2-(2-methoxyethoxy) acetic acid, and in synthesis of B4, polyoxa fattyacid was used to replace 2-(2-methoxyethoxy) acetic acid. According tomass spectrum (MS) detection results, the mass-to-charge ratios of B2and B3 are 1926 and 2454, respectively, which are consistent to theircalculated molecular weights, 1926.11 and 2454.74. According toMatrix-Assisted Laser Desorption/Ionization Time of Flight MassSpectrometry (MALDI-TOF-MS), B4's molecular weight is about 14964, whichis consistent with its calculated molecular weight, 14964.56, as shownin Table 31.

TABLE 31 Character, mass spectrum and fluorescence test results of B1-B4No. n Character Molecular weight by MS Fluorescence B1 1 White powder1573 None B2 5 White powder 1926 None B3 11 White powder 2454 None B4150 White powder 14964 None

Example 37: Synthesis of B10-B24

B10-B24 were synthesized by a similar method for B1, except that theamino acids used for linking are different, as shown in Table 32.

Corresponding R₂ amino acid and R₃ amino acid were dissolved inN,N-dimethylformamide, respectively. The condensating agent, such as1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride, was addedand reactions were allowed to take place at 0-25□ for 0.5-2 hours. ThenAsn was added and reaction was taken place at 0-25□ for 2-24 hours. Thereaction solution was purified to obtain a tripeptide. The tripeptidewas used to replace Ala-Ala-Asn as an intermediate to prepare B10-B24according to Example 36. Molecular weights of B10-B24, as detected bymass spectrum, are shown in the following table, which are consistent totheir respective calculated molecular weights.

TABLE 32 Character and mass spectrum results of B10-B24 MolecularCalculated No. R₂ R₃ Character weight by MS molecular weight B10 Ala ThrWhite powder 1604 1603.72 B11 Ala Val White powder 1602 1601.67 B12 AlaAsn White powder 1617 1616.64 B13 Thr Ala White powder 1604 1603.72 B14Thr Thr White powder 1634 1633.74 B15 Thr Val White powder 1632 1631.77B16 Thr Asn White powder 1647 1646.74 B17 Val Ala White powder 16021601.74 B18 Val Thr White powder 1632 1631.77 B19 Val Val White powder1630 1629.80 B20 Val Asn White powder 1645 1644.77 B21 Ile Ala Whitepowder 1616 1615.77 B22 Ile Thr White powder 1646 1645.80 B23 Ile ValWhite powder 1644 1643.83 B24 Ile Asn White powder 1659 1658.80

Example 38: Effect of Different Groups in the Water-Soluble Docetaxelfor Targeted Activation in Tumor Microenvironment on the Formulation ofthe Drug

B1, B2, B3 and B4 and various control compounds were dried under vacuum,sterilized via gas sterilization, and separately packing in a sterileroom. Before animal test, B1, B2, B3 and B4 were dissolved by solvent 1(injectable water) or solvent 2 (30% alcohol, 70% injectable water) anddiluted by injectable water to the desired concentration in sterileroom. On the contrary, comparative compounds (C1′, C2′, C3′, C4′, C5′,and C6′) did not satisfy the formulating requirement, as shown in Table33. Docetaxel is insoluble in water, but its solubility is significantlychanged after modification, with increased solubility in water. Changein solubility may greatly affect the formulation scheme of a drug. Ascompared to the traditional Docetaxel which is insoluble in water, B1,B2, B3 and B4 can be used to produce a soluble formulation. Thus, theirinjection doses and efficacies can be improved and auxiliary materialsthat cause allergy generally used for Docetaxel can be avoided. This isa great progress in drug development, and indicates that thewater-soluble Docetaxel for targeted activation in tumormicroenvironment has a promising innovation and prospect of use.

TABLE 33 Solutility test of the screened drugs and Effect of absence ofsimilar components in control compounds or linkage to Docetaxel at its7- or 2-position (i.e., linking the group to the OH at 7- or 2-positionof Docetaxel) on the solubility of the drugs Compounds Solvent 1 Solvent2 C1′: AAN -group 2-Docetaxel (linking at 2-position) insolubleinsoluble C2′: group 1- AANL -Docetaxel insoluble insoluble (linking at2-position) C3′: AAN -Docetaxel (linking at 2-position) insolubleinsoluble C4′: group 1- AAN -group 2-Docetaxel insoluble insoluble(linking at 7-position) C5′: group 1- AANL -group 2-Docetaxel insolublesoluble (linking at 2-position) C6′: group 1- AANK -group 2-Docetaxelinsoluble insoluble (linking at 2-position) B1 insoluble soluble B2soluble soluble B3 soluble soluble B4 soluble soluble

Group 1 and group 2 mentioned below are identical to the above group 1and 2, respectively.

In Table 33, AAN, AANL and AANK indicate the linkage formed by a smallpeptide in the compounds, A is Ala, N is Asn, L is Leu and K is Lys.

The water-soluble Docetaxel derivatives for targeted activation in tumormicroenvironment of the present disclosure were based on a great amountof synthetic experiments. In these experiments, we designed a lot ofcomplicated compounds having different linking manners. Then thecomplicated compounds were linked to position 2 or 7 of Docetaxel, thatis, they were linked to Docetaxel via the OH at position 2 or position7. The resultant Docetaxel derivatives were screened through activationefficiency in the presence of tumor tissue or aspartate endopeptidase.The screened derivatives were further screened through inhibition oftumor for R₂, R₃ and n. The activated site that is specific to the tumortissue locates between AAN and group 2. After cleaving by activation,group 2 can be freely released, thereby releasing Docetaxel. Because theactive center of asparagine endopeptidase locates at the bottom of itsglobular depression and the cleavage site should be close to the activecenter, it is very important if there is a steric hindrance to thecleavage site produced by the complicated compound.

According to the screening results, it is presumed that linking of group2 may effectively avoid steric hindrance produced by directly linkingDocetaxel, which thereby not affecting approach of asparagineendopeptidase. And, the structure-efficacy of group 1 may increase thepolarity of the cleavage site, which allows the more water-solubleprotease to be easily to approach the cleavage site and thereby toincrease the cleaving efficiency. Linking to position 2 of Docetaxelcould obviously reduce steric hindrance produced by Docetaxel toprotease, expose more groups, each of which as a whole is hydrophilic,and increase cleaving efficiency and water solubility.

Example 39: Methods for Determining the Contents of B1, B2, B3 and B4and their Content Ranges

As detected by analytic HPLC (Agilent 1220 series, C8 column 5 μm, 4.6mm ID×250 mm; the mobile phase is 0-95% acetonitrile (ACN)), thepurities of B1, B2, B3 and B4 are all in the range of 95-99%.

Example 40: Various Effects of Different Groups in Present Water-SolubleDocetaxel Derivatives for Targeted Activation in Tumor Microenvironmenton the Activation of Paclitaxel Drugs in Tumor Tissue

The mutual structure-efficacy of Docetaxel with the groups linkeddetermined the targeting and activation effects in tissues. At 37□,sample compounds were added into 100 μg acidized tumor tissuehomogenates in a concentration of 1 mg/ml. The enzyme in tumor tissuehomogenates could release Docetaxel. Reduction of compounds and increaseof Docetaxel were detected by HPLC, thereby comparing the activationefficiency of the drugs by the tumor tissue.

TABLE 34 Activation ratio (%) of B1, B2, B3 and B4 in homogenates fromdifferent tumor tissues Cells producing B1 activation B2 activation B3activation B4 activation Different tumor tissues tumor efficiency (%)efficiency (%) efficiency (%) efficiency (%) Human fibrosarcoma HT-108079.1 79.9 71.6 78.6 Human breast cancer MDA-MB435 93.5 90.3 95.1 89.9Human ovarian cancer SK-OV-3 93.1 90.2 84.3 67.6 Human colon cancerHT-29 83.9 94.6 96.4 95.3 Human chronic K562 69.0 77.7 74.6 78.6leukemia Human pancreatic Panc-1 86.9 90.6 90.4 89.8 cancer Humannon-small cell A549 91.2 94.1 86.0 87.8 lung cancer Human prostatecancer PC-3 76.1 86.7 83.9 83.0 Human liver cancer Hepg2 76.3 89.2 88.178.4 Human renal cancer OS-RC-2 90.9 90.5 91.1 88.4 Human heart / 12.28.3 2.5 4.4

TABLE 35 Effect of changes of similar components in control compounds orlinkage to Docetaxel at its 7- or 2-position on activation efficiency ofthe drugs by MDA-MB231 tumor tissue activation Compounds efficiency (%)C1′: AAN -group 2- Docetaxel (linking at 2-position) 17.5 C2′: group 1-AANL - Docetaxel (linking at 2-position) 46.6 C3′: AAN - Docetaxel(linking at 2-position) 38.5 C4′: group 1- AAN -group 2- Docetaxel 16.3(linking at 7-position) C5′: group 1- AANL -group 2- Docetaxel 67.4(linking at 2-position) C6′: group 1- AANK -group 2- Docetaxel 56.6(linking at 2-position) B1 93.4 B2 91.6 B3 90.6 B4 88.5

As table 35 shows, activation efficiency of linkage to Docetaxel at its2-position is far higher than that at 7-position.

According to the results, different groups in the present Docetaxel fortargeted activation in tumor microenvironment have various effects onthe activation of Docetaxel drugs in tumor tissue. The mutualstructure-efficacy of Docetaxel with the groups linked determined thetargeting and activation effects in tissues. Activation of B1, B2, B3and B4 in different tumor types (10 kinds) proved their broad treatmentspectrum (Table 36). Meanwhile, certain compounds produced in thescreening were compared, and the activation efficiency in the same humanbreast cancer MDA-MB435 tissue was examined. It was proved that therespective group selection in B1, B2, B3 and B4 had relatively higheractivation efficiency (Table 36).

Example 41: Detection of Maximum Tolerated Dose (MTD) by IntravenousInjection of the Drugs

Test purpose: to investigate the acute toxicity of the present newformulations via detecting MTD by intravenous injection.

Test drugs: B1, B2, B3 and B4 injections, diluted to correspondingconcentrations by physiological saline when testing.

Animal: the first class BALB/C mice, weighing 19-21 g and all mice beingfemale.

Method and results: 210 BALB/C mice were randomly divided into 21 groupsaccording to their body weights, with 10 mice in each group. As shown inTable 37, the mice were intravenously injected with B1, B2, B3 and B4for just one time in a dose of 0 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg,and 200 mg/kg. Control tests were performed by injecting 0.2 mlphysiological saline or Docetaxel. Animals were observed for 17continuous days for presence or absence of the following behaviors oneach day: pilo-erection, hair tousle and lackluster, lethargy, stoop andirritable reaction, and body weight and death were recorded. Bloodsamples were taken on the 3, 5 and 14 days for counting the whole bloodcells. Animals were anatomized on day 14 to take the heart, liver,kidney, lung, spleen, and pancreas for HE staining.

TABLE 37 Comparison of mortality rates of test mice receiving differentdoses of B1, B2, B3 and B4 injections, physiological saline or Docetaxelinjection Number Dose Number of of dead Mortality rate Group (mg/kg)animal animal (%) 1 physiological  0 mg/kg 10 0 0 saline 2 B1 125 mg/kg10 0 0 3 B1 150 mg/kg 10 0 0 4 B1 175 mg/kg 10 0 0 5 B1 200 mg/kg 10 220 6 B2 125 mg/kg 10 0 0 7 B2 150 mg/kg 10 0 0 8 B2 175 mg/kg 10 0 0 9B2 200 mg/kg 10 2 20 10 B3 125 mg/kg 10 0 0 11 B3 150 mg/kg 10 0 0 12 B3175 mg/kg 10 0 0 13 B3 200 mg/kg 10 3 3 14 B4 125 mg/kg 10 0 0 15 B4 150mg/kg 10 0 0 16 B4 175 mg/kg 10 0 0 17 B4 200 mg/kg 10 1 10 18 Docetaxel 25 mg/kg 10 0 0 19 Docetaxel  30 mg/kg 10 3 30% 20 Docetaxel  35 mg/kg10 6 60% 21 Docetaxel  40 mg/kg 10 10 100% 

Results and discussions: no pilo-erection, hair tousle and lackluster,lethargy, stoop, irritable reaction and death were observed in micereceiving 175 mg/kg B1, B2, B3 and B4 injections. As shown in Table 37,the MTD of the B1 and B2 injections were about 150 mg/kg, which is farbeyond the MTD of Docetaxel, 25 mg/kg. The MTD for intravenousadministration of a test drug is an important reference index for drugtoxicity. The results indicate that the toxicity of the Docetaxelreleased by targeted activation is significantly reduced as comparedwith Docetaxel.

Example 42: Study on Efficacy of the Present B1, B2, B3 and B4Injections in Nude Mice

Test purpose: to investigate the anti-tumor efficacy of B1, B2, B3 andB4 in mice model for tumor treatment.

Test drug: B1, B2, B3 and B4 injections and Docetaxel injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Human prostate cancer PC-3 cells were purchased from American typeculture collection (ATCC) and identified according the specificationprovided by ATCC. Cells were cultivated in DMEM culture solutioncontaining 10% fetal bovine serum at 37° C. and 5% CO₂. The cells werepassaged for every three days and cells within the 15th passage wereused.

2) Production of tumor. 5×10⁶Panc-1 cells were subcutaneously injectedto the back of the nude mice. Mice were randomly grouped after the tumorreached at least 100 mm³. Then treatment began and the day on which thetreatment began was day 1.

3) Course of treatment

According to the clinical application of B1, B2, B3 and B4, drugs wereintravenously injected (IV). B1, B2, B3 and B4 were administered in adose of less than ⅙ MTD, i.e., 25 mg/kg, and Docetaxel was administeredin a dose of ⅓ MTD, i.e., 8.3 mg/kg. The control group was administeredby physiological saline. Drugs were administered once weekly for fourweeks.

4) Grouping and test results are shown in Table 38.

TABLE 38 Effect of B1, B2, B3 and B4, Docetaxel and control group ontumor treatment in nude mice inhibitory rate Number of Size of tumor(mm³) on tumor Group animal Day 10 Day 24 Day 10 Day 24 B1 group 10 92.4 ± 59.66 128.45 ± 105.56 67.8 66.3 B2 group 10 67.35 ± 53.67 136.45± 57.45  76.5 64.2 B3 group 10 89.45 ± 78.67 178.45 ± 79.45  68.8 53.1B4 group 10 68.88 ± 35.56 215.67 ± 103.45 76.0 43.4 Docetaxel 10 254.75± 146.55 263.65 ± 184.67 11.1 30.7 treatment group Control group 10286.64 ± 214.45 684.25 ± 324.45 / /

5) Results and discussions: As shown in Table 38, inhibition on tumorgrowth by B1, B2, B3 and B4 were greatly improved as compared with thegroups treating by Docetaxel using the same molar concentration and thecontrol group.

Example 43: Study on Efficacy of B1, B2, B3 and B4 in D121 Tumor ImmuneModel

Test purpose: to investigate the anti-tumor efficacy of B1, B2, B3 andB4 in a D121 lung cancer model for immune treatment.

Test drugs: B1, B2, B3, B4, and Docetaxel were administered in a dose of13.2 μmol/kg, and the dose of PDL1-antibody was 5 μg/kg.

Animal: C57 mice of 6-8 weeks old, all female.

Production of Tumor Model:

1) D121 lung tumor cells were purchased from ATCC. Cells were cultivatedin DMEM culture solution containing 10% fetal bovine serum at 37° C. and5% CO₂. The cells were passaged for every three days and cells withinthe 15th passage were used.

2) Tumor immunization. 5×10⁵ D121 lung cancer cells (purchased fromATCC) which were killed by irradiation were intraperitoneally injectedto mice. The mice were injected for 3 times, once every two weeks. Afterimmunization, mice were injected with tumor cells and the drugs wereadministered weekly for 4 weeks.

3) Production of tumor. At day 32, 10⁶ live lung tumor cells weresubcutaneously injected to the back of the C57 mice immunized by tumor.Treatment began when the tumor grew to 0.3-0.4 cm.

4) Analysis on tumor CD8+ T cells. The tumor tissue was homogenated andindividual cells in the tumor were filtered, separated and washed bybuffer twice, then cultivated with the leucocyte common antigen CD45-PEand T-lymphocyte antigen CD8-FITC marked antibodies for 1 hour atambient temperature. The cells were washed by phosphate buffercontaining 1% fetal bovine serum twice and then analyzed for the ratioof the T lymphocyte antigen (CD8) positive cells in the leucocyte commonantigen (CD45) positive cells by flow cytometry. Increasement of theratio indicates increased T lymphocyte cells and thus the animalimmunity against the tumor was improved.

5) Grouping and test results are shown in Table 39.

TABLE 39 Effect on inhibition of tumor and immune activation of B1, B2,B3 and B4, Docetaxel and control inhibitory rate on Number of Size oftumor (mm³) tumor % Group animal Day 18 Day 18 CD8: CD45 (%) Immunegroup, without 8 1937.45 ± 368.45 / 4.6 D121 dead tumor cells Immunegroup (Control 8 1620.39 ± 389.23 13.4 group) Immune group + B1 8 271.36 ± 157.56 83.25 18.9 Immune group + B2 8 375.727 ± 301.67 76.8117.4 Immune group + B3 8 350.393 ± 124.65 78.37 17.8 Immune group + B4 8324.005 ± 155.56 80.00 16.6 Immune group + B1 + PDL1 8  71.28 ± 35.5995.60 23.6 antibody Immune group + Docetaxel 8 1242.30 ± 359.48 23.335.4 Immune group + Docetaxel + 8 1068.39 ± 451.16 34.06 7.1 PDL1antibody

6) Results and discussion. As shown in table 39, treatment effects ofB1, B2, B3 and B4 on C57 mice were greatly improved as compared to thecontrol group and the other treatment groups. B1 and PDL1-antibody showan excellent synergistic effect in promoting immunization and treatment.They can inhibit tumor growth via improving immunization.

Example 44: Study on Efficacy of B1, B2, B3 and B4 in BALB/C Mice Modelfor Tumor Metastasis

Test purpose: to investigate the anti-tumor efficacy of B1, B2, B3 andB4 in BALB/C mice model for treatment of tumor metastasis.

Test drug: B1, B2, B3 and B4 injections and Docetaxel injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: BALB/C mice of 6-8 weeks old, all female.

2. Production of tumor model

1) 4T1 cells were purchased from American type culture collection (ATCC)and identified according the specification provided by ATCC. Cells werecultivated in DMEM culture solution containing 10% fetal bovine serum at37° C. and 5% CO₂. The cells were passaged for every three days andcells within the 15th passage were used.

2) Production of tumor metastasis. 10⁶ T1 cells were subcutaneouslyinjected to the back of the BALB/C mice. Mice were randomly groupedafter the tumor grew to about 1.5 cm. The subcutaneous tumor was removedby surgery and drug treatment began. Mice were killed after anesthesiaon day 27. The whole lung was taken out and put into Bouin's solutionfor staining. The number of the tumor metastasized to lung was countedwith anatomical microscope.

3) Course of treatment

According to the clinical application of B1, B2, B3 and B4, drugs wereintravenously injected (IV). B1, B2, B3 and B4 were administered in adose of ⅙ MTD, i.e., 17.6 μmol/kg, and Docetaxel was administered in adose of ⅙ MTD, i.e., 3 μmol/kg. The control group was administered byphysiological saline. Drugs were administered once for every three daysfor 4 times.

4) Grouping and test results are shown in Table 40.

TABLE 40 Effects of B1, B2, B3 and B4, Docetaxel and control oninhibition of tumor metastasis in nude BALB/C mice Number of Number ofmetastasized Inhibitory rate on Group animal tumor metastasis B1 Group10 5 ± 3 96.0 B2 Group 10 11 ± 7  91.3 B3 Group 10 17 ± 11 86.5 B4 Group10 18 ± 16 85.7 Docetaxel 10 85 ± 17 32.5 treatment group Control group10 126 ± 37  /

5) Results and discussion. As shown in Table 40, the inhibitory effecton tumor metastasis of BALB/C mice was greatly improved afterintraperitoneal injection of B1, B2, B3 and B4, as compared with theDocetaxel group and the control group, indicating that this kind ofdrugs exhibits an excellent efficacy on anti-tumor metastasis.

Example 45: Study on Efficacy of B1 Injection in Multiple Tumor Models

Test purpose: to investigate the anti-tumor spectrum of B1 throughmultiple tumor models from mice

Test drug: B1 injection, diluted to corresponding concentrations byphysiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Corresponding tumor cells were purchased from American type culturecollection (ATCC) and identified according the specification provided byATCC. Cells were cultivated in DMEM culture solution containing 10%fetal bovine serum at 37° C. and 5% CO₂. The cells were passaged forevery three days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶ corresponding cells were subcutaneouslyinjected to the back of the nude mice. Mice were randomly grouped afterthe tumor reached at least 100 mm³. Then treatment began and the day onwhich the treatment began was day 1.

3) Course of treatment. According to the clinical application of B1, B1was administered in a dose of ⅙ MTD, i.v., 17.6 μmol/kg. The controlgroup was administered by physiological saline. Animals wereadministered once weekly for three weeks.

4) Grouping and test results are shown in Table 41.

TABLE 41 Treatment effect of B1 in multiple tumor models inhibitory rateon Group Tumor cell tumor (Day 26) Human breast cancer MDA-MB435 78.84%Human ovarian cancer SK-OV-3 74.67% Human colon cancer HT-29 74.56%Human chronic leukemia K562 72.56% Human colon caner HT1080 84.46% Humanpancreatic cancer Panc-1 73.56% Human non-small cell lung cancer A54974.56% Human liver cancer Hepg2 81.56% Human renal cancer OS-RC-2 86.67%

5) Results and discussion. As shown in Table 41, B1 shows an excellentefficacy in multiple tumor models, demonstrating that the anti-tumordrug has a wide anti-tumor spectrum.

In other examples of the present disclosure, activation efficiency,inhibitory rate on tumor and inhibitory rate on metastasis of thepresent water-soluble Docetaxel derivatives (B10˜B24) for targetedactivation with different amino acid structures were examined usingmethods same as that in example 40, 42 and 44. Results were showed intable 42.

TABLE 42 activation efficiency, inhibitory rate on tumor and onmetastasis of B10-B24 inhibitory activation rate on inhibitory Compoundefficiency tumor (%) rate on No. R₂ R₃ n (%) (Day 38) metastasis (%) B10Ala Thr 5 66.18 65.66 77.55 B11 Ala Val 5 44.05 47.77 46.01 B12 Ala Asn5 39.71 50.56 84.37 B13 Thr Ala 5 78.27 63.07 90.37 B14 Thr Thr 5 38.7854.18 30.40 B15 Thr Val 5 56.46 47.56 40.64 B16 Thr Asn 5 34.33 70.6258.73 B17 Val Ala 5 31.64 60.28 67.00 B18 Val Thr 5 68.04 72.17 82.82B19 Val Val 5 39.81 57.08 70.62 B20 Val Asn 5 44.98 49.43 73.83 B21 IleAla 5 51.29 44.88 66.07 B22 Ile Thr 5 72.28 61.52 72.90 B23 Ile Val 559.46 67.42 47.05 B24 Ile Asn 5 50.67 49.09 56.04

Results and discussion. As shown in Table 42, compounds B10˜B24 could beactivated and had some effects on inhibition of tumor growth and onmetastasis, indicating the screening of inventors could optimize theactivation and treatment of tumor. It should be understood that theabove descriptions of preferred Examples are not intended to limit thesubject invention. After reading the above details, it is apparent tothe skilled artisan that amino acids at position R₂ and R₃ of thepresent drugs or compounds can be changed or replaced.

In some examples of the invention, other water-soluble Docetaxelderivatives for targeted activation in tumor microenvironment weresynthesized, of which n is any integer between 1-150, R₂ is Ala, Thr,Val or Ile; R₃ is Ala, Thr, Val or Asn. And they were subjected toformulation test as done in Examples 38, MTD test as done in Example 41,study on efficacy on tumor as done in Examples 42 and 43, study onefficacy of inhibiting metastasis as done in Example 44 and study onefficacy on multiple tumors as done in Example 45. Similar results toB1-B4 were obtained. As demonstrated by the experiments, when n is inthe range of 1-150, the inhibitory rate on tumor is slightly reduced asn increases. The activation activity also slightly decreases and mass ofdrugs in the same mole increases, as n increases. However, the metabolichalf life of the drug also increases as n increases. Therefore, theentire efficacy is only slightly decreased and when n is in the range of1-150, all compounds could produce similar technical effect to B1-B4.

Example 46: Synthesis of Docetaxel Derivatives for Targeted Activationin Tumor Microenvironment

Step 1: Synthesis of Cbz-L-Ala-L-Ala-OMe(Carboxybenzyl-L-Ala-L-Ala-methyl ester) (I)

N-Carboxybenzyl-L-Ala (N-Cbz-L-Ala) (100 g, 0.45 mol) was dissolved inN,N-dimethylformamide (3 L). 1-hydroxylbenzotriazole (72.6 g, 0.54 mol)and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (103.3 g,0.54 mol) were added when stirring. After reaction for 1 hour, Themixture was cooled to 0□ and L-Ala methyl ester (46.2 g, 0.45 mol) andN,N-diisopropyl ethylamine (173.8 g, 1.34 mol) in N,N-dimethylformamide(1 L) were added when stirring and then the resultant mixture wasstirred at ambient temperature for 10 hours. The solvents were removedby evaporation under reduced pressure. The crude product was dissolvedin dichloromethane (2 L), washed subsequently by saturated ammoniumchloride solution, water and saturated sodium chloride solution. Theorganic phase was dried by anhydrous sodium sulphate. After removing thesolvents by evaporation under reduced pressure, the crude product wasrecrystallized to obtain a white solid I (101 g, Yield 73.1%).

Step 2: Synthesis of Cbz-L-Ala-L-Ala-OH (II)

Cbz-L-Ala-L-Ala-OMe (100 g, 0.34 mol) prepared in step 1) was dissolvedin a mixed solution of tetrahydrofuran (2 L) and water (1 L). Aftercooling to 0□, 1M lithium hydroxide solution (400 mL) were added. Theresultant mixture was stirred for reaction for 10 hours. Concentratedhydrochloric acid was dropped to adjust the pH to be less than 6 andtetrahydrofuran were removed by rotary evaporation. The residual waterphase was extracted by dichloromethane (1 L×3). The organic phase wasdried by anhydrous sodium sulphate. A white solid II was obtained aftervaporizing and drying under reduced pressure (88 g; Yield, 92.2%).

Step 3: Synthesis of Fmoc-L-Asn (Trt)-L-4-amino benzyl alcohol (III)

Fmoc-L-Asn (Trt)-OH (fluorenylmethoxycarbonyl-triphenylmethyl-L-Asn) (20g, 0.03 mol), 2-(7-azabenzotriazol)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (15 g, 0.04 mol) and DMF (200 mL) were addedinto a three-neck flask and stirred for 30 minutes. After cooling to 0°C., a solution of 4-amino benzyl alcohol (4.1 g, 0.03 mol) in DMF (5mL), and N,N-diisopropyl ethylamine (8.7 g, 0.06 mol) were separatelyadded. The resultant mixture was stirred at ambient temperature for 3hours. Then Most of DMF were removed by rotary evaporation. The residuewas dissolved in ethyl acetate (200 mL), washed subsequently bysaturated ammonium chloride solution and saturated sodium chloridesolution and dried by anhydrous sodium sulphate. After filtration, thesolvent was removed by evaporation. The resultant crude product waspulping to obtain a white solid III (21.3 g, Yield 90%).

Step 4: Synthesis of L-Asn (Trt)-L-4-amino benzyl alcohol (IV)

Fmoc-L-Asn (Trt)-L-4-amino benzyl alcohol (13.0 g, 18 mmol) prepared instep 3) was dissolved in N,N-dimethylformamide (80 mL). Piperidine (30mL) was added and then stirred at ambient temperature for 2 hours. Thesolvents were removed by evaporation under reduced pressure. And theresultant product was dried under high vacuum within a vacuum dryingoven to remove a small quantity of piperidine. A pale yellow solid IVwas obtained, which could be use in the next step without purification.

Step 5: Synthesis of Cbz-L-Ala-L-Ala-L-Asn (Trt)-4-amino benzyl alcohol(V)

Cbz-L-Ala-L-Ala-OH (6.0 g, 20.4 mmol) prepared in step 2),benzotriazol-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU,11.6 g, 30.6 mmol) and DMF (50 mL) were added into a three-neck flaskand stirred for 30 minutes in an ice bath. A solution of L-Asn(Trt)-4-amino benzyl alcohol in DMF (50 mL), andN,N-diisopropylethylamine (7.89 g, 61.2 mmol) were added separatelyunder 0° C. The resultant mixture was stirred overnight at ambienttemperature. The solvents were removed by evaporation under reducedpressure. The residue was dissolved in acetyl acetate (200 mL), washedsubsequently by saturated ammonium chloride solution and saturatedsodium chloride solution and dried by anhydrous sodium sulphate. Afterfiltration, the solvent was removed by evaporation. The resultant crudeproduct was recrystallized to obtain a white solid V (15 g, Yield 97%).

Step 6: Synthesis of L-Ala-L-Ala-L-Asn (Trt)-4-amino benzyl alcohol (VI)

Cbz-L-Ala-L-Ala-L-Asn(Trt)-4-amino benzyl alcohol (5.0 g, 6.61 mmol)prepared in step 5) were dissolved in THF (150 mL). 10% Pd/C (1 g) wasadded. After introducing hydrogen gas, the resultant mixture was stirredfor reaction under normal temperature and normal pressure for 5 hours.Pd/C was removed by filtration and washed by methanol. The filtrates andthe washing solutions were pooled. Most solvents were removed by rotaryevaporation to obtain a crude product. After column chromatography, awhite solid VI was obtained (2.0 g, Yield 49%).

Step 7: Synthesis of 2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn(Trt)-4-amino benzyl alcohol (VII)

2-(2-methoxyethoxy) acetic acid (432 mg, 3.22 mmol) were dissolved inN,N-dimethylformamide (20 mL). Benzotriazol-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (1.83 g, 4.83 mmol) were added and stirred for 30minutes. Then L-Ala-L-Ala-L-Asn (Trt)-4-amino benzyl alcohol (2.0 g,3.22 mmol) prepared in step 6) and N,N-diisopropylethylamine (1.24 g,9.61 mmol) in N,N-dimethylformamide (20 mL) were dropped into theresultant mixture. After dropping, the temperature was slowly raised toambient temperature and then the mixture was stirred for 10 hours. Mostof DMF were removed by evaporation under reduced pressure. The residuewas dissolved in acetyl acetate (200 mL), washed subsequently bysaturated ammonium chloride solution and saturated sodium chloridesolution and dried by anhydrous sodium sulphate. After filtration, thesolvent was removed by rotary evaporation. The resultant crude productwas purified by silica gel column chromatography to obtain a white solidVII (1.2 g, Yield 50%).

Step 8: Synthesis of 2-(2-methoxyethoxy)acetyl-L-Ala-L-Ala-L-Asn-4-amino benzyl alcohol (VIII)

2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn (Trt)-4-amino benzylalcohol (1.0 g, 1.36 mmol) prepared in step 7) were dissolved indichloromethane (10 mL). Trifluoroacetic acid (2 mL) were added and thenthe resultant mixture was stirred at ambient temperature for 5 hours.The reaction solution was washed by water and separated. The organicphase was dried by anhydrous sodium sulphate and the solvents wereremoved by evaporation under reduced pressure. The residualtrifluoroacetic acid was removed by evaporation under high vacuum. Theresultant crude product was purified by column chromatography to obtainX (600 mg, Yield 89%).

Step 9: Synthesis of 2-(2-methoxyethoxy)acetyl-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenyl carbonate (IX)

A solution of 2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn-4-aminobenzyl alcohol (500 mg, 1.01 mmol) in dichloromethane (10 mL) was addedinto a three-neck flask. P-nitrophenyl chloroformate (406 mg, 2.02 mmol)and pyridine (160 mg, 2.03 mmol) in a dichloromethane solution weresubsequently dropped into the mixture in an ice bath under protection bynitrogen gas. After dropping, the resultant mixture was stirred atambient temperature overnight. The reaction solution was washed by waterand separated. The organic phase was dried by anhydrous sodium sulphateand the solvents were removed by rotary evaporation. The resultant crudeproduct was purified by column chromatography to obtain IX (450 mg,Yield 67%).

Step 10: Synthesis of 2-(2-methoxyethoxy)acetyl-L-Ala-L-Ala-L-Asn-4-amino benzyl-Docetaxel (C1)

2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenylcarbonate (880 mg, 1.3 mmol) prepared in step 9) and Docetaxel (1.3 g,1.6 mmol) were dissolved by anhydrous N,N-dimethylformamide (20 mL) andcooled to 0□. DMAP (326 mg, 2.6 mmol) were added and then stirred atambient temperature overnight. The reaction solution was poured intodichloromethane. The organic phases were pooled, washed by water, driedby anhydrous sodium sulphate. The solvents were removed by rotaryevaporation to obtain a crude product. The crude product was purified bycolumn chromatography to obtain the target product D1 (340 mg, Yield49.2%).

D2, D3 and D4 were synthesized by making reference to D1, except thatthe acetic acids substituted by alkoxy group used in step 7 havedifferent molecular weights. When synthesizing D2, 3, 6, 9, 12, 15,18-hexaoxanonadecanoic acid was used to replace 2-(2-methoxyethoxy)acetic acid, in synthesis of D3, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36-dodecaoxaheptatriacontanoic acid was used to replace2-(2-methoxyethoxy) acetic acid, and in synthesis of D4, polyoxa fattyacid was used to replace 2-(2-methoxyethoxy) acetic acid. According tomass spectrum (MS) detection results, the mass-to-charge ratios of D1,D2 and D3 are 1329, 1505, and 1770, respectively, which are consistentto their calculated molecular weights, 1329.40, 1505.61, and 1769.93.According to Matrix-Assisted Laser Desorption/Ionization Time of FlightMass Spectrometry (MALDI-TOF-MS), D4's molecular weight is about 14497,which is consistent with its calculated molecular weight, 14497.31, asshown in Table 43.

TABLE 43 Character, mass spectrum and fluorescence test results of C1-C4No. n Character Molecular weight by mass spectrum fluorescence Output(milligram) Yield D1 1 White powder 1329 None 340 49.2%   D2 5 Whitepowder 1505 None 157 49% D3 11 White powder 1770 None 365 46% D4 300White powder 14497 None 345 28%

Example 47: Effect of Different Groups in the Docetaxel for TargetedActivation in Tumor Microenvironment on the Formulation of the Drug

Different groups in the Docetaxel for targeted activation in tumormicroenvironment show great effect on the formulation of the drug. D1,D2, D3, D4 and various control compounds were dried under vacuum,sterilized via gas sterilization, and separately packing in a sterileroom. Before animal test, D1, D2, D3 and D4 were dissolved by solvent 1(injectable water) or solvent 2 (45% alcohol, 55% injectable water) anddiluted by injectable water to the desired concentration in sterileroom. On the contrary, comparative compounds (C1′, C2′, C3′, C4′, C5′,and C6′) did not satisfy the formulating requirement, as shown in Table44. Docetaxel is insoluble in water, but its solubility is significantlychanged after modification, with increased solubility in water. Changein solubility may greatly affect the formulation scheme of a drug. Ascompared to the traditional Docetaxel which is insoluble in water, D1,D2, D3 and D4 can be used to produce a soluble formulation. Thus, theirinjection doses and efficacies can be improved and auxiliary materialsthat cause allergy generally used for Docetaxel can be avoided. This isa great progress in drug development, and indicates that the Docetaxelfor targeted activation in tumor microenvironment has a promisinginnovation and prospect of use.

TABLE 44 Effect of absence of similar components in control compounds orlinkage to Docetaxel at its 7- or 2-position (i.e., linking the group tothe OH at 7- or 2-position of Docetaxel) on the solubility of the drugCompound Solvent 1 Solvent 1 C1′: AAN -group 2-Docetaxel (linking at2-position) insoluble insoluble C2′: group 1- AANL -Docetaxel insolubleinsoluble (linking at 2-position) C3′: AAN -Docetaxel (linking at2-position) insoluble insoluble C4′: group 1- AAN -group 2-Docetaxelinsoluble insoluble (linking at 7-position) C5′: group 1- AANL -group2-Docetaxel insoluble soluble (linking at 2-position) C6′: group 1- AANK-group 2-Docetaxel insoluble insoluble (linking at 2-position) D1insoluble soluble D2 insoluble soluble D3 soluble soluble D4 solublesoluble

Group 1 and group 2 mentioned below are identical to the above group 1and 2, respectively.

In Table 44, AAN, AANL and AANK indicate the linkage formed by a smallpeptide in the compounds, A is Ala, N is Asn, L is Leu and K is Lys.

Group 1 in the Docetaxel for targeted activation in tumormicroenvironment is significantly important for the activation andefficacy of the entire drug. When group 1 is absent, the solubility andactivation efficiency are greatly affected.

Group 2 in the Docetaxel for targeted activation in tumormicroenvironment is significantly important for the activation andefficacy of the entire drug. When group 2 is absent, the activationefficiency and the blockage of toxicity are greatly affected.

The Docetaxel derivatives for targeted activation in tumormicroenvironment of the present disclosure were based on a great amountof synthetic experiments. In these experiments, we designed a lot ofcomplicated compounds having different linking manners. Then thecomplicated compounds were linked to position 2 or 7 of Docetaxel, thatis, they were linked to Docetaxel via the OH at position 2 or position7. The resultant Docetaxel derivatives were screened through activationefficiency in the presence of tumor tissue or aspartate endopeptidase.The screened derivatives were further screened through inhibition oftumor for R₂, R₃ and n. The activated site that is specific to the tumortissue locates between AAN and group 2. After cleaving by activation,group 2 can be freely released, thereby releasing Docetaxel. Because theactive center of asparagine endopeptidase locates at the bottom of itsglobular depression and the cleavage site should be close to the activecenter, it is very important if there is a steric hindrance to thecleavage site produced by the complicated compound.

According to the screening results, it is presumed that linking of group2 may effectively avoid steric hindrance produced by directly linkingDocetaxel, which thereby not affecting approach of asparagineendopeptidase. And, the structure-efficacy of group 1 may increase thepolarity of the cleavage site, which allows the more water-solubleprotease to be easily to approach the cleavage site and thereby toincrease the cleaving efficiency. Linking to position 2 of Docetaxelcould obviously reduce steric hindrance produced by Docetaxel toprotease, expose more groups, each of which as a whole is hydrophilic,and increase cleaving efficiency and water solubility.

Example 48: Methods for Determining the Contents of D1, D2, D3 and D4 inRespective Products and their Content Ranges

As detected by analytic HPLC (Agilent 1220 series, C8 column 5 μm, 4.6mm ID×250 mm; the mobile phase is 0-95% acetonitrile (ACN)), thepurities of D1, D2, D3 and D4 are all in the range of 95-99%.

Example 49: Various Effects of Different Groups in Present DocetaxelDerivatives for Targeted Activation in Tumor Microenvironment on theActivation of Paclitaxel Drugs in Tumor Tissue

Different groups in present Docetaxel derivatives for targetedactivation in tumor microenvironment have different effects on theactivation of Paclitaxel drugs in tumor tissue. The mutualstructure-efficacy of Docetaxel with the groups linked determined thetargeting and activation effects in tissues. In the experiments, at 37□,compounds were added into proteases in 100 μg acidized tumor tissuehomogenates in a concentration of 1 mg/ml. The tumor tissue homogenatescould release Docetaxel. Reduction of compound and increase of Docetaxelwere detected by HPLC, thereby comparing the activation efficiency ofthe drug by the tumor tissue. It was found that the linker linking tothe screening compound exhibited highest activation efficiency.Activation in different tumor types also indicates that the drugs have abroad treatment spectrum (table 45). Meanwhile certain compoundsproduced in the screening were compared and their activation efficiencyin same tissue was analyzed. It is proved the chemical group selectionfor D1 has the highest activation efficiency (table 45), and theactivation efficiency of D2˜D4 in different tumor tissue homogenates isclose to D1.

TABLE 45 Activation ratio (%) of D1, D2, D3 and D4 in homogenates fromdifferent tumor tissues Activation ratio (%) in homogenates fromdifferent Cells tumor tissues Different tumor tissues producing tumor D1D2 D3 D4 Human fibrosarcoma HT-1080 77.23 67.86 71.11 67.14 Human breastcancer MDA-MB435 83.07 82.26 81.36 83.52 Human ovarian cancer SK-OV-379.56 86.14 71.37 57.42 Human colon cancer HT-29 71.46 80.91 82.26 81.54Human chronic leukemia K562 68.23 65.97 63.18 66.78 Human pancreaticcancer Panc-1 85.32 84.42 82.35 83.79 Human non-small cell A549 77.7680.46 83.26 75.24 lung cancer Human prostate cancer PC-3 87.57 88.5686.67 84.15 Human liver cancer Hepg2 85.77 76.14 75.15 66.78 Human renalcancer OS-RC-2 77.76 82.35 77.76 81.45

TABLE 46 Effect of changes of similar but different components incontrol compounds or linkage to Docetaxel at its 7- or 2-position onactivation efficiency of the drugs by MDA-MB231 tumor tissue activationCompounds efficiency(%) C1′: AAN-group 2-Docetaxel 23.2 (linking at2-position) C2′: group 1-AANL-Docetaxel 50.4 (linking at 2-position)C3′: AAN-Docetaxel 34.4 (linking at 2-position) C4′: group 1-AAN-group2-Docetaxel 16.8 (linking at 7-position) C5′: group 1-AANL-group2-Docetaxel 39.7 (linking at 2-position) C6′: group 1-AANK-group2-Docetaxel 57.4 (linking at 2-position) D1 91.5 D2 91.1 D3 90.8 D4 89.5

As table 46 shows, activation efficiency of linkage to Docetaxel at its2-position is higher than that at 7-position.

According to the results, different groups in the present Docetaxel fortargeted activation in tumor microenvironment have various effects onthe activation of Docetaxel drugs in tumor tissue. The mutualstructure-efficacy of Docetaxel with the groups linked determined thetargeting and activation effects in tissues.

Example 50: Detection of Maximum Tolerated Dose (MTD) by IntravenousInjection of the Test Drugs

Test purpose: to investigate the acute toxicity of the new drugformulations via detecting MTD by intravenous injection.

Test drugs: D1, D2, D3 and D4 injections, diluted to correspondingconcentrations by physiological saline when testing.

Animal: the first class BALB/C mice, weighing 19-21 g and all mice beingfemale.

Method and results: 210 BALB/C mice were randomly divided into 21 groupsaccording to their body weights, with 10 mice in each group. As shown inTable 47, the mice were intravenously injected with D1, D2, D3 and D4for just one time in a dose of 0 mg/kg, 125 mg/kg, 150 mg/kg, 175 mg/kg,and 200 mg/kg. Control tests were performed by injecting 0.2 mlphysiological saline or Docetaxel. Animals were observed for 17continuous days for presence or absence of the following behaviors oneach day: pilo-erection, hair tousle and lackluster, lethargy, stoop andirritable reaction, and body weight and death were recorded. Bloodsamples were taken on the 3, 5 and 14 days for counting the whole bloodcells. Animals were anatomized on day 14 to take the heart, liver,kidney, lung, spleen, and pancreas for HE staining.

TABLE 47 Comparison of mortality rates of test mice receiving differentdoses of D1, D2, D3 and D4 injections, physiological saline or Docetaxelinjection Number Mortality Dose Number of dead rate Group injections(mg/kg) of animal animal (%) 1 physiological  0 mg/kg 10 0 0 saline 2 D1125 mg/kg 10 0 0 3 D1 150 mg/kg 10 0 0 4 D1 175 mg/kg 10 0 0 5 D1 200mg/kg 10 1 10 6 D2 125 mg/kg 10 0 0 7 D2 150 mg/kg 10 0 0 8 D2 175 mg/kg10 0 0 9 D2 200 mg/kg 10 2 20 10 D3 125 mg/kg 10 0 0 11 D3 150 mg/kg 100 0 12 D3 175 mg/kg 10 0 0 13 D3 200 mg/kg 10 1 10 14 D4 125 mg/kg 10 00 15 D4 150 mg/kg 10 0 0 16 D4 175 mg/kg 10 0 0 17 D4 200 mg/kg 10 0 1018 Docetaxel  25 mg/kg 10 0 0 19 Docetaxel  30 mg/kg 10 2 20% 20Docetaxel  35 mg/kg 10 5 50% 21 Docetaxel  40 mg/kg 10 10 100% 

Results and discussions: no pilo-erection, hair tousle and lackluster,lethargy, stoop, irritable reaction and death were observed in micereceiving 150 mg/kg D1, D2, D3 and D4 injections. As shown in Table 47,the MTD of the D1 and D2 injections were about 150 mg/kg, which is farbeyond the MTD of Docetaxel, 25 mg/kg. The MTD for intravenousadministration of a test drug is an important reference index for drugtoxicity. The results indicate that the toxicity of the Docetaxelreleased by targeted activation is significantly reduced as comparedwith Docetaxel.

Example 51: Study on Efficacy of D1, D2, D3 and D4 Injections in NudeMice

Test purpose: to investigate the anti-tumor efficacy of D1, D2, D3 andD4 in mice model for tumor treatment.

Test drug: D1, D2, D3 and D4 injections and Docetaxel injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Human prostate cancer PC-3 cells were purchased from American typeculture collection (ATCC) and identified according the specificationprovided by ATCC. Cells were cultivated in DMEM culture solutioncontaining 10% fetal bovine serum at 37° C. and 5% CO₂. The cells werepassaged for every three days and cells within the 15th passage wereused.

2) Production of tumor. 5×10⁶PC-3 cells were subcutaneously injected tothe back of the nude mice. Mice were randomly grouped after the tumorreached at least 100 mm³. Then treatment began and the day on which thetreatment began was day 1.

3) Course of treatment

According to the clinical application of D1, D2, D3 and D4, drugs wereintravenously injected (IV). D1, D2, D3 and D4 were administered in adose of less than ⅙ MTD, i.e., 25 mg/kg, and Docetaxel was administeredin a dose of ⅓ MTD, i.e., 8.3 mg/kg. The control group was administeredby physiological saline. Drugs were administered once weekly for fourweeks.

4) Grouping and test results are shown in Table 48.

TABLE 48 Effect of E1, E2, E3, E4, Docetaxel and control group on tumortreatment in nude mice Size of tumor inhibitory rate Number (mm³) ontumor Group of animal Day 10 Day 24 Day 10 Day 24 D1 group 10 86.45 ±26.42 143.34 ± 44.42 75.0 80.5 D2 group 10 78.53 ± 36.89 113.52 ± 41.8877.3 84.5 D3 group 10 67.43 ± 28.93 157.45 ± 64.74 80.5 78.6 D4 group 1078.56 ± 36.74 167.33 ± 63.65 77.3 77.2 Docetaxel 10 168.66 ± 79.43  313.75 ± 157.42 51.3 57.3 treatment group Control group 10  346.4 ±121.78  734.45 ± 216.56 / /

5) Results and discussions: As shown in Table 48, inhibition on tumorgrowth by D1, D2, D3 and D4 were greatly improved as compared with thegroups treating by Docetaxel using the same molar concentration and thecontrol group.

Example 52: Study on Efficacy of D1, D2, D3 and D4 in D121 Tumor ImmuneModel

Test purpose: to investigate the anti-tumor efficacy of D1, D2, D3 andD4 in a D121 lung cancer model for immune treatment.

Test drug: D1, D2, D3, D4 and Docetaxel, all used in 13.2 μmol/kg; PDL1antibody, 5 μg/kg.

Animal: C57 mice of 6-8 weeks old, all female.

Production of Tumor Model:

1) D121 lung tumor cells were purchased from ATCC. Cells were cultivatedin DMEM culture solution containing 10% fetal bovine serum at 37° C. and5% CO₂. The cells were passaged for every three days and cells withinthe 15th passage were used.

2) Tumor immunization. 5×10⁵ D121 lung cancer cells (purchased fromATCC) which were killed by irradiation were intraperitoneally injectedto mice. The mice were injected for 3 times, once every two weeks. Afterimmunization, mice were injected with tumor cells and the drugs wereadministered weekly for 4 weeks.

3) Production of tumor. At day 32, 10⁶ live D121 lung tumor cells weresubcutaneously injected to the back of the C57 mice immunized by tumor.Treatment began when the tumor grew to 0.3-0.4 cm.

4) Analysis on tumor CD8+ T cells. The tumor tissue was homogenated andindividual cells in the tumor were filtered, separated and washed bybuffer twice, then cultivated with the leucocyte common antigen CD45-PEand T-lymphocyte antigen CD8-FITC marked antibodies for 1 hour atambient temperature. The cells were washed by phosphate buffercontaining 1% fetal bovine serum twice and then analyzed for the ratioof the T lymphocyte antigen (CD8) positive cells in the leucocyte commonantigen (CD45) positive cells by flow cytometry. Increasement of theratio indicates increased T lymphocyte cells and thus the animalimmunity against the tumor was improved.

5) Grouping and test results are shown in Table 49.

TABLE 49 Effect on inhibition of tumor and immune activation of D1, D2,D3, D4, Docetaxel and control Size inhibitory Number of tumor rate onCD8: of (mm³) tumor % CD45 Group animal Day 18 Day 18 (%) Immune group,without 8 1673.56 6.4 D121 dead tumor cells Immune group 8 1425.56 12.6(Control group) Immune group + D1 8 324.45 77.2 18.5 Immune group + D2 8312.43 78.1 17.3 Immune group + D3 8 323.56 77.3 17.7 Immune group + D48 246.85 82.7 16.3 Immune group + D1 + 8 136.43 90.4 23.6 PDL1 antibodyImmune group + 30 1268.64 11.0 6.9 Docetaxel Immune group + 8 846.6740.6 9.4 Docetaxel + PDL1 antibody

6) Results and discussion. Treatment effects of D1, D2, D3 and D4 on C57mice were greatly improved as compared to the control group and theother treatment groups. D1 and PDL1-antibody show an excellentsynergistic effect in promoting immunization and treatment. They caninhibit tumor growth via improving immunization.

Example 53: Study on Efficacy of D1, D2, D3 and D4 in BALB/C Mice Modelfor Tumor Metastasis

Test purpose: to investigate the anti-tumor efficacy of D1, D2, D3 andD4 in BALB/C mice model for treatment of tumor metastasis.

Test drug: D1, D2, D3 and D4 injections and Docetaxel injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: BALB/C mice of 6-8 weeks old, all female.

2. Production of tumor model

1) 4T1 cells were purchased from American type culture collection (ATCC)and identified according the specification provided by ATCC. Cells werecultivated in DMEM culture solution containing 10% fetal bovine serum at37° C. and 5% CO₂. The cells were passaged for every three days andcells within the 15th passage were used.

2) Production of tumor metastasis. 10⁶ T1 cells were subcutaneouslyinjected to the back of the BALB/C mice. Mice were randomly groupedafter the tumor grew to about 1.5 cm. The subcutaneous tumor was removedby surgery and drug treatment began. Mice were killed after anesthesiaon day 27. The whole lung was taken out and put into Bouin's solutionfor staining. The number of the tumor metastasized to lung was countedwith anatomical microscope.

3) Course of treatment

According to the clinical application of D1, D2, D3 and D4, drugs wereintravenously injected (IV). D1, D2, D3 and D4 were administered in adose of ⅙ MTD, i.e., 25 mg/kg, and Docetaxel was administered in a doseof ⅙ MTD, i.e., 4.2 mg/kg. The control group was administered byphysiological saline. Drugs were administered once for every three daysfor 4 times.

4) Grouping and test results are shown in Table 50.

TABLE 50 Effects of D1, D2, D3, D4, Docetaxel and control on inhibitionof tumor metastasis in BALB/C mice Number of Inhibitory Numbermetastasized rate on Group of animal tumor metastasis D1 Group 10  5 ± 395.2 D2 Group 10 13 ± 8 97.3 D3 Group 10  17 ± 13 93.0 D4 Group 10  19 ±13 90.8 Docetaxel 10 156 ± 24 89.7 treatment group Control group 10 185± 35 /

5) Results and discussion. As shown in Table 50, the inhibitory effecton tumor metastasis of BALB/C mice was greatly improved afterintraperitoneal injection of D1, D2, D3 and D4, as compared with theDocetaxel group and the control group, indicating that this kind ofdrugs exhibits an excellent efficacy on anti-tumor metastasis.

Example 54: Study on Efficacy of C1 in Multiple Tumor Models

Test purpose: to investigate the anti-tumor spectrum of C1 throughmultiple tumor models from mice

Test drug: C1 injection, diluted to corresponding concentrations byphysiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Corresponding tumor cells were purchased from American type culturecollection (ATCC) and identified according the specification provided byATCC. Cells were cultivated in DMEM culture solution containing 10%fetal bovine serum at 37° C. and 5% CO₂. The cells were passaged forevery three days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶ corresponding cells were subcutaneouslyinjected to the back of the nude mice. Mice were randomly grouped afterthe tumor reached at least 100 mm³. Then treatment began and the day onwhich the treatment began was day 1.

3) Course of treatment. According to the clinical application of D1, D1was administered in a dose of ⅙ MTD, i.e., 25 mg/kg. The control groupwas administered by physiological saline. Animals were administered onceweekly for three weeks.

4) Grouping and test results are shown in Table 51.

TABLE 51 Treatment effect of D1 in multiple tumor models inhibitory rateGroup Tumor cell on tumor (Day 26) Human breast cancer MDA-MB435 93.5%Human ovarian cancer SK-OV-3 82.9% Human colon cancer HT-29 68.6% Humanchronic leukemia K562 84.6% Human colon caner HT1080 94.6% Humanpancreatic cancer Panc-1 89.4% Human non-small cell lung cancer A54990.4% Human liver cancer Hepg2 75.7% Human renal cancer OS-RC-2 87.7%

5) Results and discussion. As shown in Table 51, D1 shows an excellentefficacy in multiple tumor models, demonstrating that the drug has awide anti-tumor spectrum.

Compounds D10-D24 were also prepared in the present disclosure bysimilar method for synthesizing D1, except that the starting amino acidsused for linking were different, as shown in Table 52. Corresponding R₂amino acid and R₃ amino acid were dissolved in N,N-dimethylformamide.The same condensating agent, 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride, was added respectively and reactions wereallowed to take place at 0-25° C. for 0.5-2 hours. Then Asn was addedand reaction was taken place at 0-25° C. for 2-24 hours to obtain atripeptide. Molecular weights of D10-D24 (n=1), as detected by massspectrum (MS), are shown in Table 47, which are consistent to theirrespective calculated molecular weights.

Activation property, inhibitory rate on tumor and inhibitory rate onmetastasis of Docetaxel for targeted activation in tumormicroenvironment having different amino acid structures were tested bythe same methods as described in Examples 49, 51 and 53. The results areshown in Table 47. Since results from Examples 49, 51 and 53 indicatethat n is preferably in the range of 1-11, at which range the drugs havethe same treatment effects, n in D10-D24 is selected as 1 except that R₂and R₃ are different.

TABLE 52 Activation property, inhibitory rate on tumor and inhibitoryrate on metastasis of D10-D24 for targeted activation in tumormicroenvironment inhibitory inhibitory No. Molecular rate on rate on ofweight Calculated activation tumor meta- Com- Char- by molecularefficiency (%) stasis pound R₂ R₃ acter MS weight (%) (Day 38) (%) D10Ala Thr White 1360 1359.72 65.4% 65.6% 75.3% powder D11 Ala Val White1358 1357.67 42.6% 46.2% 44.5% powder D12 Ala Asn White 1373 1372.6438.4% 49.5% 81.6% powder D13 Thr Ala White 1360 1359.72 75.7% 61.3%87.4% powder D14 Thr Thr White 1390 1389.74 37.5% 52.4% 29.4% powder D15Thr Val White 1388 1387.77 54.6% 45.8% 39.3% powder D16 Thr Asn White1403 1402.74 33.2% 68.3% 56.8% powder D17 Val Ala White 1358 1357.7430.6% 58.3% 64.8% powder D18 Val Thr White 1388 1387.77 65.8% 69.8%80.1% powder D19 Val Val White 1386 1385.80 38.5% 55.2% 68.3% powder D20Val Asn White 1401 1400.77 43.5% 47.8% 71.4% powder D21 Ile Ala White1372 1371.77 49.6% 43.4% 63.9% powder D22 Ile Thr White 1402 1401.8069.9% 59.5% 70.5% powder D23 Ile Val White 1400 1399.83 57.5% 65.2%45.5% powder D24 Ile Asn White 1415 1414.80   49% 47.48% 54.2% powder

Results and discussion: As shown in Table 52, compounds D10˜D24 could beactivated and had some effects on inhibition of tumor growth and onmetastasis, indicating the screening of inventors could optimize theactivation and treatment of tumor.

In some other examples of the invention, other Docetaxel derivatives fortargeted activation in tumor microenvironment were synthesized, of whichn is any integer between 1-300, R₂ is Ala, Thr, Val or Ile; R₃ is Ala,Thr, Val or Asn. And they were subjected to formulation test, MTD test,study on efficacy on tumor, study on efficacy of inhibiting metastasisand study on efficacy on multiple tumors. Similar results to D1-D4 wereobtained. As demonstrated by the experiments, when n is in the range of1-300, the inhibitory rate on tumor is slightly reduced as n increases.The activation activity also slightly decreases and mass of drugs in thesame mole increases, as n increases. However, the metabolic half life ofthe drug also increases as n increases. Therefore, the entire efficacyis only slightly decreased and when n is in the range of 1-300, allcompounds could produce similar technical effect to D1-D4.

Example 55: Synthesis of Mitomycin Targeting to TumorMicroenvironment 1) Synthesis of Fmoc-L-Ala-L-Ala-OMe(fluorenylmethoxycarbonyl-L-Ala-L-Ala-methyl ester) (I)

Fmoc-L-Ala-OH (fluorenylmethoxycarbonyl-L-Ala) (33 g, 0.1 mol) wasdissolved in N,N-dimethylformamide (1 L). A solution of1-hydroxylbenzotriazole (20.2 g, 0.15 mol),1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (34 g, 0.15mol) and L-Ala methyl ester (13.9 g, 0.1 mol) and N,N-diisopropylethylamine (25.8 g, 0.2 mol) in N,N-dimethylformamide (500 mL) wereadded when stirring and then the resultant mixture was stirred atambient temperature for 10 hours. The solvents were removed byevaporation under reduced pressure. The crude product was dissolved indichloromethane (2 L), washed subsequently by saturated ammoniumchloride solution, water and saturated sodium chloride solution. Theorganic phase was dried by anhydrous sodium sulphate. The organic phasewas dried by anhydrous sodium sulphate. After removing the solvents byevaporation under reduced pressure, the crude product was recrystallizedto obtain a white solid I (30 g, Yield 75.1%).

2) Synthesis of Fmoc-L-Ala-L-Ala-OH(fluorenylmethoxycarbonyl-L-Ala-L-Ala) (II)

Fmoc-L-Ala-L-Ala-OMe (40 g, 0.1 mol) was dissolved in a mixed solutionof tetrahydrofuran (2 L) and water (1 L). After cooling, 1M lithiumhydroxide solution (400 mL) were added. The resultant mixture wasstirred for reaction for 10 hours. Concentrated hydrochloric acid wasdropped to adjust the pH to be less than 6 and tetrahydrofuran wereremoved by evaporation under reduced pressure. The residual water phasewas extracted by dichloromethane (1 L×3). The organic phase was dried byanhydrous sodium sulphate. A white solid II was obtained aftervaporizing and drying under reduced pressure (36 g; Yield, 94%).

3) Synthesis of Fmoc-L-Asn (Trt)-L-4-amino benzyl alcohol (III)

Fmoc-L-Asn (Trt)-OH (fluorenylmethoxycarbonyl-triphenylmethyl-L-Asn) (20g, 0.03 mol), 2-(7-azabenzotriazol)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) (15 g, 0.04 mol) and DMF (200 mL) were addedinto a three-neck flask and stirred for 30 minutes. A solution of4-amino benzyl alcohol (4.1 g, 0.03 mol) in DMF (5 mL), andN,N-diisopropyl ethylamine (8.7 g, 0.06 mol) were separately added. Theresultant mixture was stirred at ambient temperature for 3 hours. Thenthe solvents were removed by evaporation under reduced pressure. Theresidue was dissolved in ethyl acetate (200 mL), washed subsequently bysaturated ammonium chloride solution and saturated sodium chloridesolution and dried by anhydrous sodium sulphate. After filtration, thesolvent was removed by evaporation. The resultant crude product waspulping to obtain a white solid III (21.3 g, Yield 90%).

4) Synthesis of L-Asn (Trt)-L-4-amino benzyl alcohol (IV)

Fmoc-L-Asn (Trt)-L-4-amino benzyl alcohol (13 g, 18 mmol) was dissolvedin N,N-dimethylformamide (80 mL). Piperidine (30 mL) was added and thenstirred at ambient temperature for 2 hours. The solvents were removed byevaporation under reduced pressure. And the resultant product was driedunder high vacuum within a vacuum drying oven to remove a small quantityof piperidine. A pale yellow solid IV was obtained, which could be usein the next step without purification.

5) Synthesis of Fmoc-L-Ala-L-Ala-L-Asn (Trt)-4-amino benzyl alcohol (V)

Fmoc-L-Ala-L-Ala-OH (5.4 g, 14 mmol),benzotriazol-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU, 8g, 21 mmol) and DMF (50 mL) were added into a three-neck flask andstirred for 30 minutes in an ice bath under protection by nitrogen gas.A solution of L-Asn (Trt)-4-amino benzyl alcohol (6.7 g, 14 mmol) in DMF(50 mL), and N,N-diisopropylethylamine (5.5 g, 42 mmol) were addedseparately under 0° C. The resultant mixture was stirred overnight atambient temperature. The solvents were removed by evaporation underreduced pressure. The residue was dissolved in acetyl acetate (200 mL),washed subsequently by saturated ammonium chloride solution andsaturated sodium chloride solution and dried by anhydrous sodiumsulphate. After filtration, the solvent was removed by evaporation. Theresultant crude product was pulped to obtain a white solid V (18.5 g,Yield 78%).

6) Synthesis of L-Ala-L-Ala-L-Asn (Trt)-4-amino benzyl alcohol (VI)

Fmoc-L-Asn (Trt)-L-4-amino benzyl alcohol (864 mg, 1 mmol) weredissolved in N,N-dimethylformamide (30 mL). Piperidine (10 mL) was addedand then stirred at ambient temperature for 2 hours. The solvents wereremoved by evaporation under reduced pressure. A pale yellow solid IVwas obtained, which could be use in the next step without purification.

7) Synthesis of 2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn(Trt)-4-amino benzyl alcohol (VII)

2-(2-methoxyethoxy) acetic acid (134 mg, 1 mmol) were dissolved inN,N-dimethylformamide (5 mL). After cooling to 0° C.,3-(Diethoxyphosphoryloxy)-1, 2, 3-benzotrizin-4-one (DEPBT, 450 mg, 1.5mmol) were added and stirred for 30 minutes. Then L-Ala-L-Ala-L-Asn(Trt)-4-amino benzyl alcohol (621 mg, 1 mmol) and N,N-diisopropylethylamine (387 mg, 3 mmol) were added. The reaction temperature wasslowly raised to ambient temperature in the dark and then stirred for 5hours. The reaction solution was poured into 200 mL aqueous acetic acidsolution and extracted by dichloromethane. The organic phases werepooled, washed by water and dried by anhydrous sodium sulphate. Thesolvents were removed by evaporation under reduced pressure to obtain anorange red crude product. The crude product was purified by silica gelcolumn chromatography to obtain a white powder VII (479 mg, Yield 65%).

8) Synthesis of 2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn(Trt)-4-aminobenzyl-4-nitrophenyl carbonate (VIII)

2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn (Trt)-4-amino benzylalcohol (1.9 g, 2.6 mmol) were added into a three-neck flask anddissolved in dichloromethane (10 mL). A solution of 4-nitrophenylchloroformate (1 g, 5.2 mmol) and pyridine (400 mg, 5.2 mmol) indichloromethane were dropped. The resultant mixture was stirred atambient temperature overnight. The reaction solution was washed by waterand separated. The organic phase was dried by anhydrous sodium sulphate.The solvents were removed by evaporation under reduced pressure. Thecrude product was purified by silica gel column chromatography to obtainVIII (1.8 g, Yield 80%).

9) Synthesis of 2-(2-methoxyethoxy)acetyl-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenyl carbonate (IX)

2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn(Trt)-4-aminobenzyl-4-nitrophenyl carbonate was dissolved indichloromethane (2 mL). Trifluoroacetic acid (2 mL) were added and thenstirred at ambient temperature for 2 hours. The reaction solution waswashed by water and separated. The organic phase was dried by anhydroussodium sulphate. The solvents were removed by evaporation under reducedpressure. The crude product was purified by column chromatography toobtain IX (625 mg, Yield 47%).

10) Synthesis of 2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn-4-aminobenzyl mitomycin (E1)

2-(2-methoxyethoxy) acetyl-L-Ala-L-Ala-L-Asn-4-aminobenzyl-4-nitrophenylcarbonate (400 mg, 0.6 mmol) was dissolved in N,N-dimethylformamide (10mL). Mitomycin C (200 mg, 0.6 mmol), 1-hydroxy benzotriazole (HOBT, 17mg, 0.12 mmol) and N,N-diisopropyl ethylamine (156 mg, 1.2 mmol) wereadded. The temperature was raised to ambient temperature and then theresultant mixture was stirred 10 hours. The solvents were removed byevaporation under reduced pressure. The residue was dissolved indichloromethane (200 mL), washed subsequently by saturated ammoniumchloride solution and saturated sodium chloride solution and dried byanhydrous sodium sulphate. After filtration, the solvent was removed byevaporation. The resultant crude product was purified by columnchromatography to obtain a pale yellow solid, which was the targetcompound E1 (237 mg, Yield 46%).

E2, E3 and E4 were synthesized by making reference to E1, except thatthe acetic acids substituted by alkoxy group used in step 7 havedifferent molecular weights. When synthesizing E2, 3, 6, 9, 12, 15,18-hexaoxanonadecanoic acid was used to replace 2-(2-methoxyethoxy)acetic acid, in synthesis of E3, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30,33, 36-dodecaoxaheptatriacontanoic acid was used to replace2-(2-methoxyethoxy) acetic acid, and in synthesis of E4, polyoxa fattyacid was used to replace 2-(2-methoxyethoxy) acetic acid. According tomass spectrum (MS) detection results, the mass-to-charge ratios of E1,E2 and E3 are 855, 1032, and 1296, respectively, which are consistent totheir calculated molecular weights, 855.85, 1032.06, and 1296.37.According to Matrix-Assisted Laser Desorption/Ionization Time of FlightMass Spectrometry (MALDI-TOF-MS), E4's molecular weight is about 14032,which is consistent with its calculated molecular weight, 14023.76, asshown in Table 53.

TABLE 53 Character, mass spectrum and fluorescence test results of E1-E4Molecular weight No. n Character by mass spectrum fluorescence E1 1White powder 855 None E2 5 White powder 1032 None E3 11 White powder1296 None E4 150 White powder 14023 None

Example 56: Injections for E1, E2, E3 and E4

E1, E2, E3 and E4 were dried under vacuum, sterilized via gassterilization, and separately packing in a sterile room. Before animaltest, E1 was dissolved by injectable water containing 50% alcohol anddiluted by injectable water to the desired concentration. E2, E3 and E4could be directly diluted by injectable water to the desiredconcentrations.

Example 57: Methods for Determining the Contents of E1, E2, E3 and E4 inRespective Products and their Content Ranges

As detected by analytic HPLC (Agilent 1220 series, C8 column 5 μm, 4.6mm ID×250 mm; the mobile phase is 0-95% acetonitrile (ACN)), thepurities of E1, E2, E3 and E4 are all in the range of 95-99%.

Example 58: Activation of Mitomycin for Targeted Activation in TumorMicroenvironment in Different Tumor Tissues

At 37° C., compounds were added into proteases in 100 μg acidized tumortissue homogenates in a concentration of 1 mg/ml. The tumor tissuehomogenates could release mitomycin. Reduction of compound and increaseof mitomycin were detected by HPLC, thereby comparing the activationefficiency of the drug by the tumor tissue. It was found that thecurrent compounds linking to the screened compounds exhibited highestactivation efficiency. Activation in different tumor types alsoindicates that the drugs have a broad treatment spectrum. See Table 54.

TABLE 54 Activation ratio (%) of E1, E2, E3 and E4 in homogenates fromdifferent tumor tissues Activation ratio (%) Cells in homogenates fromproducing different tumor tissues Different tumor tissues tumor E1 E2 E3E4 Human fibrosarcoma HT-1080 83.6 85.7 81.3 85.4 Human breast cancerMDA-MB435 97.3 90.6 96.3 78.8 Human ovarian cancer SK-OV-3 93.5 97.698.3 91.7 Human colon cancer HT-29 94.2 96.1 98.1 93.0 Human chronicK562 74.5 68.4 61.8 62.1 leukemia Human pancreatic cancer Panc-1 89.484.6 83.1 89.7 Human non-small cell A549 97.4 96.4 89.4 84.2 lung cancerHuman prostate cancer PC-3 78.9 86.4 74.8 89.9 Human liver cancer Hepg294.6 94.8 97.8 91.5 Human renal cancer OS-RC-2 99.7 94.5 97.6 99.1

Example 59: Detection of Maximum Tolerated Dose (MTD) by IntravenousInjection of the Test Drugs

Test purpose: to investigate the acute toxicity of the new drugformulations via detecting MTD by intravenous injection.

Test drugs: E1, E2, E3 and E4 injections, diluted to correspondingconcentrations by physiological saline when testing.

Animal: the first class BALB/C mice, weighing 19-21 g and all mice beingfemale.

Method and results: 210 BALB/C mice were randomly divided into 21 groupsaccording to their body weights, with 10 mice in each group. As shown inTable 55, the mice were intravenously injected with E1, E2, E3 and E4for just one time in a dose of 0 mg/kg, 50 mg/kg, 70 mg/kg, 90 mg/kg,and 110 mg/kg. Control tests were performed by injecting 0.2 mlphysiological saline or mitomycin. Animals were observed for 17continuous days for presence or absence of the following behaviors oneach day: pilo-erection, hair tousle and lackluster, lethargy, stoop andirritable reaction, and body weight and death were recorded. Bloodsamples were taken on the 3, 5 and 14 days for counting the whole bloodcells. Animals were anatomized on day 14 to take the heart, liver,kidney, lung, spleen, and pancreas for HE staining.

TABLE 55 Comparison of mortality rates of test mice receiving differentdoses of E1, E2, E3 and E4 injections, physiological saline or mitomycininjection Number Mortality Dose Number of dead rate Group (mg/kg) ofanimal animal (%) 1 physiological  0 mg/kg 10 0 0 saline 2 E1 50 mg/kg10 0 0 3 E1 70 mg/kg 10 0 0 4 E1 90 mg/kg 10 0 0 5 E1 110 mg/kg  10 1 106 E2 50 mg/kg 10 0 0 7 E2 70 mg/kg 10 0 0 8 E2 90 mg/kg 10 0 0 9 E2 110mg/kg  10 1 10 10 E3 50 mg/kg 10 0 0 11 E3 70 mg/kg 10 0 0 12 E3 90mg/kg 10 0 0 13 E3 110 mg/kg  10 1 10 14 E4 50 mg/kg 10 0 0 15 E4 70mg/kg 10 0 0 16 E4 90 mg/kg 10 0 0 17 E4 110 mg/kg  10 0 10 18 mitomycin 6 mg/kg 10 0 0 19 mitomycin  7 mg/kg 10 1 10% 20 mitomycin  8 mg/kg 104 40% 21 mitomycin  9 mg/kg 10 9 90%

Results and discussions: no pilo-erection, hair tousle and lackluster,lethargy, stoop, irritable reaction and death were observed in micereceiving 90 mg/kg E1, E2, E3 and E4 injections. As shown in Table 55,the MTD of the E1 and E2 injections were about 90 mg/kg, which is farbeyond the MTD of mitomycin, 6 mg/kg. The MTD for intravenousadministration of a test drug is an important reference index for drugtoxicity. The results indicate that the toxicity of the mitomycinreleased by targeted activation is significantly reduced as comparedwith mitomycin.

Example 60: Study on Efficacy of E1, E2, E3 and E4 Injections on Panc-1Cells in Nude Mice

Test purpose: to investigate the anti-tumor efficacy of E1, E2, E3 andE4 in mice model for tumor treatment.

Test drug: E1, E2, E3 and E4 injections and mitomycin injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Panc-1 cells were purchased from American type culture collection(ATCC) and identified according the specification provided by ATCC.Cells were cultivated in DMEM culture solution containing 10% fetalbovine serum at 37° C. and 5% CO₂. The cells were passaged for everythree days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶Panc-1 cells were subcutaneously injectedto the back of the nude mice. Mice were randomly grouped after the tumorreached at least 100 mm³. Then treatment began and the day on which thetreatment began was day 1.

3) Course of treatment

According to the clinical application of E1, E2, E3 and E4, drugs wereintravenously injected (IV). E1, E2, E3 and E4 were administered in adose of ⅙ MTD, i.e., 15 mg/kg, and mitomycin was administered in a doseof ⅓ MTD, i.e., 2 mg/kg. The control group was administered byphysiological saline. Drugs were administered once weekly for fourweeks.

4) Grouping and test results are shown in Table 56.

TABLE 56 Effect of E1, E2, E3, E4, mitomycin and control group on tumortreatment in nude mice Size of tumor inhibitory rate Number (mm³) ontumor Group of animal Day 10 Day 24 Day 10 Day 24 E1 group 10 76.42 ±14.96 84.62 ± 45.94 35.7% 66.1% E2 group 10 60.17 ± 30.26 42.39 ± 62.2436.4% 83.01%  E3 group 10 75.60 ± 28.54 74.39 ± 48.94 49.4% 70.2% E4group 10 73.35 ± 38.46 63.99 ± 47.13 42.9% 81.5% Mitomycin 10 118.85 ±36.47  249.54 ± 95.46  7.5% 27.9% treatment group Control group 10 128.5± 16.7   346.1 ± 104.74. / /

5) Results and discussions: As shown in Table 56, inhibition on tumorgrowth by E1, E2, E3 and E4 were greatly improved as compared with thegroups treating by mitomycin using the same molar concentration and thecontrol group.

Example 61: Study on Efficacy of E1, E2, E3 and E4 Injections on HT1080Cells in Nude Mice

Test purpose: to investigate the anti-tumor efficacy of E1, E2, E3 andE4 in mice model for tumor treatment.

Test drug: E1, E2, E3 and E4 injections and mitomycin injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) HT1080 cells were purchased from American type culture collection(ATCC) and identified according the specification provided by ATCC.Cells were cultivated in DMEM culture solution containing 10% fetalbovine serum at 37° C. and 5% CO₂. The cells were passaged for everythree days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶ HT1080 cells were subcutaneously injectedto the back of the nude mice. Mice were randomly grouped after the tumorreached at least 100 mm³. Then treatment began and the day on which thetreatment began was day 1.

3) Course of treatment

According to the clinical application of E1, E2, E3 and E4, drugs wereintravenously injected (IV). E1, E2, E3 and E4 were administered in adose of ⅙ MTD, i.e., 15 mg/kg, and mitomycin was administered in a doseof ⅓ MTD, i.e., 2 mg/kg. The control group was administered byphysiological saline. Drugs were administered once weekly for fourweeks.

4) Grouping and test results are shown in Table 57.

TABLE 57 Effect of E1, E2, E3, E4, mitomycin and control group on tumortreatment in nude mice Size of tumor inhibitory rate Number (mm³) ontumor Group of animal Day 13 Day 26 Day 13 Day 26 E1 Group 10 438.15 ±47.96 331.57 ± 114.74 51.9% 78.9% E2 Group 10 378.11 ± 68.46 137.60 ±156.42 58.5% 91.3% E3 Group 10 439.82 ± 69.62 357.63 ± 194.54 51.7%77.3% E4 Group 10 426.74 ± 46.63 304.55 ± 184.53 53.2% 80.7% Mitomycin10 876.48 ± 78.29 1410.28 ± 375.46  3.7% 10.4% treatment group Controlgroup 10 910.42 ± 96.15 1574.46 ± 456.34  / /

5) Results and discussions: As shown in Table 57, inhibition on tumorgrowth by E1, E2, E3 and E4 were greatly improved as compared with thegroups treating by mitomycin using the same molar concentration and thecontrol group.

Example 62: Study on Efficacy of E1, E2, E3 and E4 in BALB/C Mice Modelfor Tumor Metastasis

Test purpose: to investigate the anti-tumor efficacy of E1, E2, E3 andE4 in BALB/C mice model for treatment of tumor metastasis.

Test drug: E1, E2, E3 and E4 injections and mitomycin injection, dilutedto corresponding concentrations by physiological saline when testing.

Method and Results:

1. Animal: BALB/C mice of 6-8 weeks old, all female.

2. Production of tumor model

1) 4T1 cells were purchased from American type culture collection (ATCC)and identified according the specification provided by ATCC. Cells werecultivated in DMEM culture solution containing 10% fetal bovine serum at37° C. and 5% CO₂. The cells were passaged for every three days andcells within the 15th passage were used.

2) Production of tumor metastasis. 10⁶ T1 cells were subcutaneouslyinjected to the back of the BALB/C mice. Mice were randomly groupedafter the tumor grew to about 1.5 cm. The subcutaneous tumor was removedby surgery and drug treatment began. Mice were killed after anesthesiaon day 27. The whole lung was taken out and put into Bouin's solutionfor staining. The number of the tumor metastasized to lung was countedwith anatomical microscope.

3) Course of treatment

According to the clinical application of E1, E2, E3 and E4, drugs wereintravenously injected (IV). E1, E2, E3 and E4 were administered in adose of ⅙ MTD, i.e., 15 mg/kg, and mitomycin was administered in a doseof ⅙ MTD, i.e., 1 mg/kg. The control group was administered byphysiological saline. Drugs were administered once for every three daysfor 4 times.

4) Grouping and test results are shown in Table 58.

TABLE 58 Effects of E1, E2, E3, E4, mitomycin and control on inhibitionof tumor metastasis in BALB/C mice Number of Inhibitory Numbermetastasized rate on Group of animal tumor metastasis E1 Group 10 2 ± 399.2% E2 Group 10 8 ± 7 94.1% E3 Group 10 13 ± 8  90.44%  E4 Group 10 15± 16 89.0% Mitomycin 10 128 ± 25   5.9% treatment group Control group 10136.0 ± 46   /

5) Results and discussion. As shown in Table 58, the inhibitory effecton tumor metastasis of BALB/C mice was greatly improved afterintraperitoneal injection of E1, E2, E3 and E4, as compared with themitomycin group and the control group, indicating that this kind ofdrugs exhibits an excellent efficacy on anti-tumor metastasis.

Example 63: Study on Efficacy of E1, E2, E3 and E4 in D121 Tumor ImmuneModel

Test purpose: to investigate the anti-tumor efficacy of E1, E2, E3 andE4 in a D121 lung cancer model for immune treatment.

Test drug: E1, E2, E3, E4 and mitomycin, all used in 13.2 μmol/kg; PDL1antibody, 5 μg/kg.

Animal: C57 mice of 6-8 weeks old, all female.

Production of Tumor Model:

1) D121 lung tumor cells were purposed from ATCC. Cells were cultivatedin DMEM culture solution containing 10% fetal bovine serum at 37° C. and5% CO₂. The cells were passaged for every three days and cells withinthe 15th passage were used.

2) Tumor immunization. 5×10⁵ D121 lung cancer cells (purchased fromATCC) which were killed by irradiation were intraperitoneally injectedto mice. The mice were injected for 3 times, once every two weeks. Afterimmunization, mice were injected with tumor cells and the drugs wereadministered weekly for 4 weeks.

3) Production of tumor. At day 32, 10⁶ live lung tumor cells weresubcutaneously injected to the back of the C57 mice immunized by tumor.Treatment began when the tumor grew to 0.3-0.4 cm.

4) Analysis on tumor CD8+ T cells. The tumor tissue was homogenated andindividual cells in the tumor were filtered, separated and washed bybuffer twice, then cultivated with the leucocyte common antigen CD45-PEand CD8-FITC marked antibodies for 1 hour at ambient temperature. Thecells were washed by phosphate buffer containing 1% fetal bovine serumtwice and then analyzed for the ratio of the T lymphocyte antigen (CD8)positive cells in the leucocyte common antigen (CD45) positive cells byflow cytometry. Increasement of the ratio indicates increased Tlymphocyte cells and thus the animal immunity against the tumor wasimproved.

5) Grouping and test results are shown in Table 59.

TABLE 59 Effect on inhibition of tumor and immune activation of E1, E2,E3, E4, mitomycin and control Size inhibitory Number of tumor rate onCD8: of (mm³) tumor % CD45 Group animal Day 18 Day 18 (%) Immune group,8 1887.56 ± 323.4  5.2 without D121 dead tumor cells Immune group 81574.46 ± 467.34  13.1 (Control group) Immune group + E1 8 237.60 ±358.57 83.27 18.4 Immune group + E2 8 331.57 ± 124.45 83.87 19.7 Immunegroup + E3 8 357.63 ± 157.32 79.55 16.3 Immune group + E4 8 304.55 ±216.47 74.85 18.4 Immune group + E1 + 8 74.78 ± 32.74 90.94 23.6 PDL1antibody Immune group + 8 1210.28 ± 368.45  28.62 6.7 mitomycin Immunegroup + 8 1334.90 ± 274.78  7.75 7.4 mitomycin + PDL1 antibody

6) Results and discussion. Treatment effects of E1, E2, E3 and E4 on C57mice were greatly improved as compared to the control group and theother treatment groups. E1 and PDL1-antibody show an excellentsynergistic effect in promoting immunization and treatment. They caninhibit tumor growth via improving immunization.

Example 64: Study on Efficacy of E1 Injection in Multiple Tumor Models

Test purpose: to investigate the anti-tumor spectrum of E1 throughmultiple tumor models from mice

Test drug: E1 injection, diluted to corresponding concentrations byphysiological saline when testing.

Method and Results:

1. Animal: nude mice of 6-8 weeks old, all female.

2. Production of tumor model

1) Corresponding tumor cells were purchased from American type culturecollection (ATCC) and identified according the specification provided byATCC. Cells were cultivated in DMEM culture solution containing 10%fetal bovine serum at 37° C. and 5% CO₂. The cells were passaged forevery three days and cells within the 15th passage were used.

2) Production of tumor. 5×10⁶ corresponding cells were subcutaneouslyinjected to the back of the nude mice. Mice were randomly grouped afterthe tumor reached at least 100 mm³. Then treatment began and the day onwhich the treatment began was day 1.

3) Course of treatment. According to the clinical application of E1, E1was administered in a dose of ⅙ MTD, i.e., 15 mg/kg. The control groupwas administered by physiological saline. Animals were administered onceweekly for three weeks.

4) Grouping and test results are shown in Table 60.

TABLE 60 Treatment effect of E1 in multiple tumor models inhibitory rateGroup Tumor cell on tumor (Day 26) Human breast cancer MDA-MB435 86.3%Human ovarian cancer SK-OV-3 84.5% Human colon cancer HT-29 86.7% Humanchronic leukemia K562 77.3% Human colon caner HT1080 95.4% Humanpancreatic cancer Panc-1 86.5% Human non-small cell lung cancer A54995.3% Human liver cancer Hepg2 85.7% Human renal cancer OS-RC-2 81.3%

5) Results and discussion. As shown in Table 60, E1 shows an excellentefficacy in multiple tumor models, demonstrating that the drug has awide anti-tumor spectrum.

Example 65: Study on Inhibition of Scar and Choroidal Neovascularization(CNV) by E1, E2, E3 and E3 Eye Drops

Animal: C57 mice of 16 weeks old, all female and 8 animals in eachgroup.

Production and treatment of scar. After fixed irradiation throughphotocoagulation by 150 mW laser, E1, E2, E3 and E4 were dropped daily.Two weeks later, eye tissues were taken from 4 animals of each group forimmunohistochemical (HE) staining. For another 4 animals, they weresubjected to fixed irradiation through photocoagulation by 50 mW laserand then to treatment. 48 hours later, their eye tissues were taken,homogenated, filtered to isolate individual cells in the scar andchoroidal neovascularization (CNV) tissues. The cells were washed bybuffer twice and stained by biotin-conjugated anti-F4/80 (biotin-labeledprecursor cell antigen from macrophage) and FITC-conjugated anti-CD45(isothiocyanate fluorescein labeled leucocyte common antigen) at ambienttemperature for 1 hour. Then the cells were washed by PBS containing 1%fetal bovine serum twice and then analyzed for the ratio of themacrophage precursor antigen positive cells in the leucocyte commonantigen (CD45) positive cells by flow cytometry. Reduction in the ratioindicates decrease of the macrophage precursor antigen positive cells,demonstrating that the macrophages associated with the disease in theanimal were inhibited. The results are shown in Table 61.

TABLE 61 Study on inhibition of scar and choroidal neovascularization(CNV) by E1, E2, E3 and E3 eye drops Maximal scar radius Number observedby pathological CDF4/80CD45 Group of animal staining (pixel) (%) Controlgroup 8 1246 ± 335  16.2 ± 3.2  E1 8 332 ± 124 7.1 ± 1.4 E2 8 348 ± 1467.7 ± 1.7 E3 8 369 ± 185 8.3 ± 2.4 E4 8 484 ± 252 9.2 ± 2.1 mitomycin 8953 ± 249 14.6 ± 2.4 

Results and discussion. E1, E2, E3 and E4 have greatly improvedtreatment effect on scar radius and inhibition of macrophage as comparedto the control group and the mitomycin group.

E10-E24 were synthesized by a similar method for E1, except that theamino acids used for linking are different, as shown in Table 62.

Specifically, corresponding R₂ amino acid and R₃ amino acid weredissolved in N,N-dimethylformamide, respectively. The condensatingagent, such as 1-ethyl-(3-dimethylaminopropyl) carbodiimidehydrochloride, was respectively added and reactions were allowed to takeplace at 0-25° C. for 0.5-2 hours. Then Asn was added and reaction wastaken place at 0-25° C. for 2-24 hours. The reaction solution waspurified to obtain a tripeptide. The tripeptide was used to replaceAla-Ala-Asn as an intermediate to prepare E10-E24 according to theprocedures of Example 55. Molecular weights of E10-E24, as detected bymass spectrum, are shown in Table 57, which are consistent to theirrespective calculated molecular weights.

TABLE 62 Character and mass spectrum results of E10-E24 MolecularCalculated No. of weight molecular Compound R₂ R₃ Character by MS weightE10 Ala Thr light blue 886 885.88 E11 Ala Val light blue 884 883.83 E12Ala Asn light blue 899 898.80 E13 Thr Ala light blue 886 885.88 E14 ThrThr light blue 916 915.90 E15 Thr Val light blue 914 913.93 E16 Thr Asnlight blue 929 928.90 E17 Val Ala light blue 884 883.90 E18 Val Thrlight blue 914 913.93 E19 Val Val light blue 912 911.96 E20 Val Asnlight blue 927 926.93 E21 Ile Ala light blue 898 897.93 E22 Ile Thrlight blue 928 927.96 E23 Ile Val light blue 926 925.99 E24 Ile Asnlight blue 941 940.96

Compounds E10-E24 were subjected to MTD test as done in Example 59,study on efficacy on tumor as done in Examples 60 and 61, study onefficacy of inhibiting metastasis as done in Example 62 and study onefficacy on multiple tumors as done in Example 64. Results show thatthey have similar results to E1-E4. As demonstrated by the experiments,when n is in the range of 1-300, the inhibitory rate on tumor isslightly reduced as n increases. The activation activity also slightlydecreases and mass of drugs in the same mole increases, as n increases.However, the metabolic half life of the drug also increases as nincreases. Therefore, the entire efficacy is only slightly decreased andwhen n is in the range of 1-300, all compounds could produce similartechnical effect to E1-E4.

Example 66: Comparison Study on Toxicity, Efficacy and ImmunologicalProperty of Legutaxel (S1′) in Tumor Model

Test purpose: to investigate the efficacy and anti-tumor immunologicalproperty of the product.

Methods and Results:

Mice were injected with Legutaxel at tail vein weekly for 3 times.According to the results of toxicity experiments observed over 21 days,no death were observed in the experiments with a dose of 140, 150 and160 mg/kg/day. Therefore, Legutaxel's dose could at least reach 160mg/kg/day during treatment.

Comparative experiments for a high dose of Legutaxel, Abraxane andPaclitaxel were performed in HT1080 model, which were used at an equalmolar dose and at an equal toxic dose. The treatment results showsignificantly different treatment efficacy. Death occurred after thethird treatment with Paclitaxel injection, as shown in FIGS. 1A and 1B.

We further studied the immunological stimulation property of Legutaxel.As demonstrated by the immunological detection of mice receiving tumortreatment, we found that the indexes for tumor-derived immunosuppressiveT cells (T reg: CD4+, CD25+, Foxp3+) obtained from the tumor-bearinggroup and in the treatment group by Paclitaxel were greatly increased.On the contrary, the index for tumor-derived immunosuppressive T cellsin treatment group by Legutaxel decreased due to targeted chemotherapy(see panels in FIGS. 3A and 3B). Meanwhile, more toxic CD8 T cells (inFIG. 2, the CD8+ positive cells are in brown, as shown by the arrows)were permeated from the tumor tissue. From this lung cancer treatmentmodel, it can be demonstrated that Legutaxel exhibits strongimmunological stimulation.

In the treatment of solid tumors, traditional chemotherapeutic drug,paclitaxel, could impair human immunity and thereby inducing drugresistance, which are crucial obstacles preventing cancer patients frombeing cured. Our experiments showed that traditional chemotherapeuticdrugs, such as paclitaxel, also greatly impair leucocyte. However,Legutaxel can only be activated in the tumor site, thus it can avoiddamage to immune system that caused by traditional chemotherapeuticdrugs. More importantly, Legutaxel could stimulate an anti-tumorimmunization, thus it can be used synergistically with immune therapy tocompletely cure cancer.

Although the contents of the invention have been detailedly introducedvia the above preferred Examples, it should be understood that the abovedescriptions are not intended to limit the subject invention. Fromexamples of S1 to S27, it can be found that the cleavage linker that isspecifically activated in tumor microenvironment and is used fortargeting a small molecule can be used to link and activate differentcompounds. Thus, it is apparent that drugs or compounds at position R₄can be changed or replaced. From the Examples in which R₁ is H, ahydrophilic group or a targeting group, it can be found that replacingor changing the group at the R₁ position is also obvious. Therefore, theprotection scope of the subject invention should be defined by theappending claims.

The invention claimed is:
 1. A compound of formula (III) or (IV)comprising a cleavable linker, wherein the cleavable linker is—R₂-R₃-Asn-4-aminobenzyl-OC(O)—:

wherein R₁ is selected from the group consisting of 6-maleimide-C₁₋₁₀alkylcarbonyl, hydroyxlaminocarbonyl-C₁₋₁₀ alkylcarbonyl, C₁₋₄alkoxyl-(C₁₋₄ alkoxyl)_(n)-C₁₋₆alkylcarbonyl, and

wherein each R is independently a C₁₋₄ alkylene, and each n isindependently any integer between 1 and 300; R₂ is an amino acid moietyselected from the group consisting of Ala, Thr, Val and Ile; R₃ is anamino acid moiety selected from the group consisting of Ala, Thr, Valand Asn; R₂ links to R₃ through an amide bond, R₃ links to Asn throughan amide bond, and Asn links to —NH— through its carbonyl; R₅ is anactive moiety of an anticancer drug containing a hydroxyl group (R₅—OH),wherein the anticancer drug is selected from the group consisting ofCamptothecin, 10-Hydroxyl Camptothecin, Topotecan, Floxuridine,5′-Deoxy-5-Fluorouridine, Cytarabine, Etoposide, Fludarabine,Capecitabine, Vincristine, Epothilone B, Paclitaxel and Docetaxel,wherein Paclitaxel and Docetaxel link to the cleavable linker throughthe hydroxyl group at position 2 when the anticancer drug is Paclitaxelor Docetaxel; R₆ is an active moiety of an anticancer drug containing anamino group (R₆—NH₂), wherein the anticancer drug is selected from thegroup consisting of Daunorubicin, Epirubicin, Methotrexate, Fludarabine,Gemcitabine, Cytarabine, Melphalan, Nimustine, Mitoxantrone, Doxorubicinand Mitomycin; the cleavable linker is characterized in that it iscleavable by contact with an asparagine endopeptidase, and the compoundis characterized by release of R₅ or R₆ through a cleavage of thecleavable linker by contact with the asparagine endopeptidase.
 2. Thecompound of claim 1, wherein R₁ is selected from the group consisting ofC₁₋₄ alkoxyl-(C₁₋₄ alkoxyl)_(n)-C₁₋₆alkylcarbonyl, and

wherein each R is independently a C₁₋₄ alkylene, and each n isindependently any integer between 1 and
 300. 3. The compound of claim 2,wherein said each n is independently any integer between 1 and
 150. 4.The compound of claim 1, wherein the compound has a structure as setforth in any of the following formulae (V), (VI), (VII), (VIII), and(IX):

wherein each n is independently any integer between 1 and 300; R₂ isAla, Thr, Val or Ile; and R₃ is Ala, Thr, Val or Asn.
 5. The compound ofclaim 4, wherein said each n is independently any integer between 1 and150.
 6. The compound of claim 1, wherein the compound is selected fromthe group consisting of:


7. The compound of claim 1, wherein the compound is selected from thegroup consisting of:

wherein, in compounds S7-S15 and S17-S18, R₁ is2-(2-methoxyethoxy)acetyl, R₂ is Thr, R₃ is Ala;

wherein, in compounds S19-S28, R₁ is 2-(2-methoxyethoxy)acetyl, R₂ andR₃ are Ala; S29-S43 represented by a formula

wherein, in compounds S29-S43, R₁ is 2-(2-methoxyethoxy)acetyl, and R₂and R₃ are shown below: No. of Compound R₂ R₃ S29 Thr Thr S30 Thr ValS31 Thr Asn S32 Val Ala S33 Val Thr S34 Val Val S35 Val Asn S36 Ile AlaS37 Ile Thr S38 Ile Val S39 Ile Asn S40 Ala Ala S41 Ala Thr S42 Ala ValS43 Ala Asn

S10′-S24′ represented by formula

in which n is 1 and R₂ and R₃ are shown as follows: No. of Compound R₂R₃ S10′ Ala Thr S11′ Ala Val S12′ Ala Asn S13′ Thr Ala S14′ Thr Thr S15′Thr Val S16′ Thr Asn S17′ Val Ala S18′ Val Thr S19′ Val Val S20′ Val AsnS21′ Ile Ala S22′ Ile Thr S23′ Ile Val S24′ Ile Asn

A10-A24 represented by formula

wherein n is 5 and R₂ and R₃ are shown in the following table: No. ofCompound R₂ R₃ A10 Ala Thr A11 Ala Val A12 Ala Asn A13 Thr Ala A14 ThrThr A15 Thr Val A16 Thr Asn A17 Val Ala A18 Val Thr A19 Val Val A20 ValAsn A21 Ile Ala A22 Ile Thr A23 Ile Val A24 Ile Asn

B10-B24 represented by

in which n, R₂ and R₃ are shown as follows: No. of Compound R₂ R₃ n B10Ala Thr 5 B11 Ala Val 5 B12 Ala Asn 5 B13 Thr Ala 5 B14 Thr Thr 5 B15Thr Val 5 B16 Thr Asn 5 B17 Val Ala 5 B18 Val Thr 5 B19 Val Val 5 B20Val Asn 5 B21 Ile Ala 5 B22 Ile Thr 5 B23 Ile Val 5 B24 Ile Asn 5

D10-D24 represented by

in which n, R₂ and R₃ are shown as follows: No. of Compound R₂ R₃ n D10Ala Thr 1 D11 Ala Val 1 D12 Ala Asn 1 D13 Thr Ala 1 D14 Thr Thr 1 D15Thr Val 1 D16 Thr Asn 1 D17 Val Ala 1 D18 Val Thr 1 D19 Val Val 1 D20Val Asn 1 D21 Ile Ala 1 D22 Ile Thr 1 D23 Ile Val 1 D24 Ile Asn 1

E10-E24 represented by formula

in which n, R₂ and R₃ are shown as follows: No. of Compound R₂ R₃ n E10Ala Thr 1 E11 Ala Val 1 E12 Ala Asn 1 E13 Thr Ala 1 E14 Thr Thr 1 EISThr Val 1 E16 Thr Asn 1 E17 Val Ala 1 E18 Val Thr 1 E19 Val Val 1 E20Val Asn 1 E21 Ile Ala 1 E22 Ile Thr 1 E23 Ile Val 1 E24 Ile Asn
 1.


8. The compound of claim 1, wherein R₂ is Thr, R₃ is Ala; or R₂ is Val,R₃ is Ala; or R₂ is Ile, R₃ is Ala; or both R₂ and R₃ are Ala.
 9. Thecompound of claim 1, wherein R₁ is 2-(2-Methoxyethoxy)acetyl,6-maleimide caproyl or N-hydroxylamino-1,8-octandioic acid-1-monoacyl.10. A pharmaceutical composition comprising the compound of claim 1 or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier or excipient.
 11. The pharmaceutical composition ofclaim 10, wherein R₁ is selected from the group consisting of C₁₋₄alkoxyl-(C₁₋₄ alkoxyl)_(n)-C₁₋₆ alkylcarbonyl, and

wherein each R is independently a C₁₋₄ alkylene, and each n isindependently any integer between 1 and
 300. 12. The pharmaceuticalcomposition of claim 11, wherein said each n is independently anyinteger between 1 and
 150. 13. The pharmaceutical composition of claim10, wherein the compound has a structure as set forth in any of thefollowing formulae (V), (VI), (VII), (VIII), and (IX):

wherein each n is independently any integer between 1 and 300; R₂ isAla, Thr, Val or Ile; and R₃ is Ala, Thr, Val or Asn.
 14. Thepharmaceutical composition of claim 13, wherein said each n isindependently any integer between 1 and
 150. 15. The pharmaceuticalcomposition of claim 10, wherein the compound is selected from the groupconsisting of:

wherein, in compounds S7-S15 and S17-S18, R₁ is2-(2-methoxyethoxy)acetyl, R₂ is Thr, R₃ is Ala;

wherein, in compounds S19-S28, R₁ is 2-(2-methoxyethoxy)acetyl, R₂ andR₃ are Ala; S29-S43 represented by a formula

wherein, in compounds S29-S43, R₁ is 2-(2-methoxyethoxy)acetyl, and R₂and R₃ are shown below: No. of Compound R₂ R₃ S29 Thr Thr S30 Thr ValS31 Thr Asn S32 Val Ala S33 Val Thr S34 Val Val S35 Val Asn S36 Ile AlaS37 Ile Thr S38 Ile Val S39 Ile Asn S40 Ala Ala S41 Ala Thr S42 Ala ValS43 Ala Asn

S10′-S24′ represented by formula

in which n is 1 and R₂ and R₃ are shown as follows: No. of Compound R₂R₃ S10′ Ala Thr S11′ Ala Val S12′ Ala Asn S13′ Thr Ala S14′ Thr Thr S15′Thr Val S16′ Thr Asn S17′ Val Ala S18′ Val Thr S19′ Val Val S20′ Val AsnS21′ Ile Ala S22′ Ile Thr S23′ Ile Val S24′ Ile Asn

A10-A24 represented by formula

wherein n is 5 and R₂ and R₃ are shown in the following table: No. ofCompound R₂ R₃ A10 Ala Thr A11 Ala Val A12 Ala Asn A13 Thr Ala A14 ThrThr A15 Thr Val A16 Thr Asn A17 Val Ala A18 Val Thr A19 Val Val A20 ValAsn A21 Ile Ala A22 Ile Thr A23 Ile Val A24 Ile Asn

B10-B24 represented by

in which n, R₂ and R₃ are shown as follows: No. of Compound R₂ R₃ n B10Ala Thr 5 B11 Ala Val 5 B12 Ala Asn 5 B13 Thr Ala 5 B14 Thr Thr 5 B15Thr Val 5 B16 Thr Asn 5 B17 Val Ala 5 B18 Val Thr 5 B19 Val Val 5 B20Val Asn 5 B21 Ile Ala 5 B22 Ile Thr 5 B23 Ile Val 5 B24 Ile Asn 5

D10-D24 represented by

in which n, R₂ and R₃ are shown as follows: No. of Compound R₂ R₃ n D10Ala Thr 1 D11 Ala Val 1 D12 Ala Asn 1 D13 Thr Ala 1 D14 Thr Thr 1 D15Thr Val 1 D16 Thr Asn 1 D17 Val Ala 1 D18 Val Thr 1 D19 Val Val 1 D20Val Asn 1 D21 Ile Ala 1 D22 Ile Thr 1 D23 Ile Val 1 D24 Ile Asn 1

E10-E24 represented by formula

in which n, R₂ and R₃ are shown as follows: No. of Compound R₂ R₃ n E10Ala Thr 1 E11 Ala Val 1 E12 Ala Asn 1 E13 Thr Ala 1 E14 Thr Thr 1 E15Thr Val 1 E16 Thr Asn 1 E17 Val Ala 1 E18 Val Thr 1 E19 Val Val 1 E20Val Asn 1 E21 Ile Ala 1 E22 Ile Thr 1 E23 Ile Val 1 E24 Ile Asn 1.